JP7226719B2 - Adeno-associated virus (AAV) delivery of anti-FAM19A5 antibodies - Google Patents

Description

Detailed description of the invention

[Technical field]
REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING , which is incorporated herein by reference in its entirety.

The present disclosure relates to recombinant adeno-associated virus (AAV) vectors and their uses. More specifically, the present disclosure relates to AAV vectors comprising nucleic acids encoding antagonists to Family, member A5 (FAM19A5) proteins with sequence similarity 19. In a particular aspect, said FAM19A5 antagonist is a single chain variable fragment that targets the FAM19A5 protein.
[Background technology]
FAM19A5 is a member of the TAFA subfamily of proteins composed of five small proteins with high homology. Tang T. Y. et al. , Genomics 83(4):727-34 (2004). These proteins contain conserved cysteine residues in place and are loosely related to macrophage inflammatory protein 1-alpha (MIP-1-alpha), a member of the CC chemokine family. Like most other TAFA proteins, FAM19A5 is predominantly expressed in specific regions of the brain and spinal cord. FAM19A5 is believed to play an important role in the development, differentiation and formation of a complete central nervous system as well as in the pathogenesis of many diseases associated with the central nervous system. Thus, in vivo administration of anti-FAM19A5 antibodies is disclosed to ameliorate symptoms associated with central nervous system injury or disease. See US Patent No. 9,579,398.

Therapeutic antibodies have been successfully used to treat a variety of diseases. Ecker D. M. , et al. , MAbs 7:9-14 (2015). However, despite the efficacy of these antibodies, they have well-known limitations that limit their widespread therapeutic use. For example, many antibodies have been described as having impaired interactions with the immune system, as well as poor pharmacokinetics and tissue accessibility. ChamesP. , et al. , Br J Pharmacol 157(2):220-223 (2009). Moreover, antibodies usually must be administered in large doses to achieve clinical efficacy, leading to increased manufacturing costs.
[Summary of Invention]
[Problems to be solved by the invention]
Accordingly, there is a need for effective and economical alternative methods for delivering therapeutic antibodies, such as anti-FAM19A5 antibodies, to subjects for disease treatment.
[Means to solve the problem]
The present disclosure provides adeno-associated virus (AAV) vectors comprising nucleic acids encoding antagonists to family, member A5 (FAM19A5) proteins with sequence similarity 19. In some embodiments, the AAV vector further comprises an intron, a signal peptide and/or one or more adeno-associated virus inverted terminal repeats (ITRs).

In some embodiments, the FAM19A5 antagonist is an anti-FAM19A5 antibody. Accordingly, in some embodiments, the AAV vector comprises nucleic acid encoding an anti-FAM19A5 antibody. In some embodiments, the anti-FAM19A5 antibody comprises Fab, Fab', F(ab')2, Fv, or single chain Fv (scFv). In certain embodiments, said anti-FAM19A5 antibody is a scFv.

In some embodiments, the AAV vector comprises codon-optimized nucleic acids. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206 or SEQ ID NO:301. In some embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, or SEQ ID NO:256.

In some embodiments, the anti-FAM19A5 antibody: (a) has the property of binding soluble human FAM19A5 with a K _D of 10 nM or less when measured by enzyme-linked immunosorbent assay (ELISA); (c) binding to membrane-bound human FAM19A5 with a K _D of 10 nM or less when measured; or (c) both (a) and (b).

In some embodiments, the anti-FAM19A5 antibody cross-competes with a reference antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 for binding to the human FAM19A5 epitope. , (i) the heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19 wherein the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:29, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:30, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:31 (ii) said heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, said heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and said heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16; wherein the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:26, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:28 (iii) said heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, said heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, and said heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; wherein the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO:23, the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO:24, and the light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:25 or (iv) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:212, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:213, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16; Said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:222, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:225, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:224.

In some embodiments, said anti-FAM19A5 antibody binds to the same FAM19A5 epitope as a reference antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, and (i) said heavy chain CDR1 The heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 18, The heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 19, The light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 19 29, wherein said light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:30, and said light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:31; (ii) said heavy chain CDR1 comprises: The heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, The heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 16, The light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO: 16 26, wherein said light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:27, and said light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:28; (iii) said heavy chain CDR1 comprises The heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13, and the light chain CDRl comprises the amino acid sequence shown in SEQ ID NO: 13. 23, wherein said light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, and said light chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; (iv) said heavy chain CDR1 comprises 212, wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 213, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 16, and said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 222 wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:225 and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:224.

In some embodiments, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope that is SEQ ID NO:6 or SEQ ID NO:9. In other embodiments, the anti-FAM19A5 antibody only binds to the FAM19A5 epitope that is SEQ ID NO:6 or SEQ ID NO:9. In certain embodiments, said anti-FAM19A5 antibody further binds to additional FAM19A5 epitopes. In some embodiments, the additional FAM19A5 epitope is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and any combination thereof.

In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein said heavy chain CDR3 is set in SEQ ID NO: 19, SEQ ID NO: 16 or SEQ ID NO: 13. including the amino acid sequence shown. In some embodiments, the heavy chain CDR1 of said anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:17, SEQ ID NO:14, SEQ ID NO:11 or SEQ ID NO:212. In some embodiments, the heavy chain CDR2 of said anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:18, SEQ ID NO:15, SEQ ID NO:12 or SEQ ID NO:213. In some embodiments, the light chain CDR1 of said anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:23 or SEQ ID NO:222. In some embodiments, the light chain CDR2 of said anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:27, SEQ ID NO:24 or SEQ ID NO:225. In certain embodiments, the light chain CDR3 of said anti-FAM19A5 antibody comprises the amino acid sequence set forth in SEQ ID NO:31, SEQ ID NO:28, SEQ ID NO:25 or SEQ ID NO:224.

In some embodiments, the anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein (i) said heavy chain CDR1, CDR2 and CDR3 are each SEQ ID NO: 17; 18 and 19, wherein said light chain CDRl, CDR2 and CDR3 comprise SEQ ID NOS: 29, 30 and 31, respectively; wherein said light chain CDRl, CDR2 and CDR3 comprise SEQ ID NOs: 26, 27 and 28, respectively; or (iv) said heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOS: 212, 213 and 16, respectively, and said light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 222, 225 and 224, respectively.

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:37, SEQ ID NO:36, SEQ ID NO:35 or SEQ ID NO:236 and/or SEQ ID NO:41, SEQ ID NO:40, SEQ ID NO:39 or It includes a light chain variable domain containing number 245. In certain embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:37 and a light chain variable domain comprising SEQ ID NO:41. In another embodiment, the anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:36 and a light chain variable domain comprising SEQ ID NO:40. In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:35 and a light chain variable domain comprising SEQ ID NO:39. In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:236 and a light chain variable domain comprising SEQ ID NO:245.

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:37, 36, 35 or 236. , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% of the same amino acid sequence. In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 41, 40, 39 or 245. , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% of the same amino acid sequence. In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:37, 36, 35 or 236. , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical amino acid sequence, said light chain variable region is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% of the amino acid sequence shown in SEQ ID NO: 41, 40, 39 or 245 , contain at least about 99% or about 100% identical amino acid sequences.

In some embodiments, the anti-FAM19A5 antibody reduces the binding of the reference antibody to human FAM19A5 by at least 10%, 20%, 30%, 40%, 50%, 60%, Inhibits 70%, 80%, 90% or 100%. In certain embodiments, the anti-FAM19A5 antibody has a concentration of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction.

The present specification also provides (a) culturing a host cell transfected with an AAV vector disclosed herein to provide a cell culture, and (b) the supernatant of said cell culture. Disclosed is a method of producing recombinant adeno-associated virus (AAV) particles comprising recovering the recombinant AAV particles from.

The present disclosure also provides a method for in vivo delivery of an antagonist to FAM19A5 protein to a subject in need thereof, comprising administering to said subject an AAV vector as described herein. do.

The disclosure also provides a method of treating a disease or condition in a subject in need thereof, comprising administering to said subject an AAV vector disclosed herein.

Also disclosed herein is a method of treating neuropathic pain in a subject in need thereof comprising administering to said subject an AAV vector of the present disclosure. In some embodiments, the neuropathic pain is peripheral neuropathic pain.

The present disclosure provides a method of increasing threshold or latency to external stimuli in a subject in need thereof, comprising administering to said subject an AAV vector disclosed herein. In some implementations, the external stimulus is a mechanical stimulus. In another embodiment, the external stimulus is thermal stimulus.

In some embodiments, the AAV vectors of the present disclosure are administered intravenously, orally, parenterally, intrathecally, intracerebroventricularly, pulmonary, intramuscularly, subcutaneously, intraperitoneally, intravitreally, epidurally, subretinal or administered intraventricularly. In some embodiments, the subject is human.
[Brief description of the drawing]
Figure 1 provides a map of the pscAAV constructs used to generate the anti-FAM19A5 scFv AAV constructs described in the Examples. As shown in Figure 1, the pscAAV construct contains the following core elements: (i) human beta globin under the CMV promoter; (ii) encoding 3-2, 1-65 or 2-13 scFv. and (iii) left-right inverted terminal repeats (ITRs).

Figures 2A, 2B and 2C show the expression levels of anti-FAM19A5 scFv after transduction of HEK293 cells with any one of the following AAV constructs: (i) scAAV8-CMV- (ii) scAAV9-CMV-3-2-ScFv; (iii) scAAV9-CMV-1-65-ScFv; or (iv) scAAV9-2-13-ScFv. In FIG. 2A, HEK293 cells were transduced with scAAV8-CMV-3-2-ScFv at two different MOIs: 5×10 ⁴ (white bars) or 1×10 ⁵ (black bars). Antibody concentrations were measured 1, 4 and 8 days after transduction. In FIG. 2B, HEK293 cells were transduced with scAAV9-CMV-3-2-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ). Antibody concentrations were measured at a single time point, 8 days after transduction. In FIG. 2C, HEK293 cells were transfected with scAAV9-CMV-1-65-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ) or scAAV9-2-13-ScFv (white bars with dots: MOI=1×10 ⁵ ; black bars with dots=2×10 ⁵ ).

Figures 3A, 3B and 3C show the mean fluorescence intensity (MFI) of human FAM19A5-Fc binding to U87MG cells in the presence or absence of supernatants of cells transduced with different anti-FAM19A5AAV constructs. ing. FIG. 3A shows data for scAAV9-CMV-1-65-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 1-65scFv. FIG. 3B shows data for scAAV9-2-13-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 2-13 scFv. FIG. 3C shows data for scAAV8-CMV-3-2-ScFv. The number "1" indicates the control group (PBS) and the number "2" indicates the 3-2 scFv.

Figures 4A and 4B show the concentration of human FAM19A5 protein in C8-D1A cell cultures after treatment with supernatants of cells transduced with different anti-FAM19A5AAV constructs. FIG. 4A compares FAM19A5 protein concentration when C8-D1A cells were cultured with the supernatant of cells transduced with scAAV8-CMV-3-2-ScFv or with PBS alone. FIG. 4B compares FAM19A5 concentration when C8-D1A cells were cultured with scAAV9-2-13-ScFv, scAAV9-CMV-1-65-ScFv or control construct (scAAV9-CMV-GFP). . In Figures 4A and 4B, "naive" represents C8-D1A cells that are not activated and produce minimal FAM19A5 protein in culture.

Figure 5 shows intracerebral anti-FAM19A5(3-2) scFv levels 1 week (upper row) and 2 weeks (lower row) after intrathecal administration of scAAV8-CMV-3-2-ScFv to C57BL/6 mice. Expression is shown. Photographs in the left column are of untreated animals (untreated). Photographs in the right column are of mice administered the AAV constructs. In the top right row of photographs, arrows denote positive staining.

Figures 6A and 6B show the effects of scAAV8-CMV-3-2-ScFv administration on mechanical allodynia (Figure 6A) and thermal hyperalgesia (Figure 6B) after peripheral nerve injury. In FIG. 6A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 6B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group.

Figures 7A and 7B show the effects of scAAV9-CMV-3-2-ScFv administration on mechanical allodynia (Figure 7A) and thermal hyperalgesia (Figure 7B) after peripheral nerve injury. In FIG. 7A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 7B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group.

Figures 8A and 8B show the effects of scAAV1-CMV-3-2-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 8A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 8B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group.

Figures 9A and 9B show the effects of scAAV9-CMV-2-13-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 9A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In Figure 9B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group.

Figures 10A and 10B show the effects of scAAV9-CMV-1-30-ScFv administration on mechanical allodynia and thermal hyperalgesia, respectively. In FIG. 10A, the effect on mechanical allodynia is expressed as the number of times animals responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 10B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group.

Figures 11A, 11B and 11C provide comparative effects of different anti-FAM19A5 scFv AAV constructs alone or in combination with other therapeutic agents. In FIG. 11A, animals were administered one of the following: (i) scAAV1-CMV-GFP (open circles); (ii) scAAV1-CMV-3-2-ScFv (filled circles); (iii). scAAV9-CMV-mIL-10 (open squares); (iv) scAAV1-CMV-3-2-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11B, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-2-13-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-2-13-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11C, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-1-30-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-1-30-ScFv+scAAV9-CMV-mIL-10 (filled squares). "*" denotes statistical significance of p<0.05 vs control group.
[Mode for carrying out the invention]
Disclosed herein are compositions, eg, adeno-associated virus (AAV) vectors, comprising nucleic acids encoding antagonists to Family, member A5 (FAM19A5) proteins with sequence similarity 19. Also provided herein are methods of using the above compositions as gene therapy methods for treating diseases or disorders, such as neuropathic pain.

To facilitate understanding of the disclosure provided herein, a number of terms and phrases are defined. Additional definitions are provided throughout the detailed description.

<I. Definition>
Throughout this disclosure, the term "a" or "a" entity is understood to refer to one or more of that entity, e.g., "an antibody" is understood to refer to one or more antibodies. As such, the terms "one" (or "a"), "one or more" and "at least one" are used interchangeably herein.

Also, as used herein, "and/or" is considered to specifically disclose each one of the two specified features or components with or without the other feature or component. should. Thus, the term "and/or" as used herein in phrases such as "A and/or B" means "A and B", "A or B", "A" (alone) and "B" intended to include (alone); Similarly, the term "and/or" used in phrases such as "A, B and/or C" is intended to include each of the following aspects: A, B and C; A or C; A or B; B or C; A and C; A and B; B and C;

It is understood that where embodiments are described herein with the term "comprising", other similar embodiments described with the terms "consisting" and/or "consisting essentially of" are also provided.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, eg, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed. ，２００２，ＣＲＣ Ｐｒｅｓｓ；Ｄｉｃｔｉｏｎａｒｙ ｏｆ Ｃｅｌｌ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，３ｒｄ ｅｄ，１９９９，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ；及びＯｘｆｏｒｄ Ｄｉｃｔｉｏｎａｒｙ Ｏｆ Ｂｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，Ｒｅｖｉｓｅｄ，２０００，Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓは、当業者に、本開示内容で使われてprovides a large number of terms.

Units, prefixes and symbols are denoted in their System International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers limiting the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

The term "about" is used herein to mean approximately, roughly, to an extent, or within that range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can vary numerical values above and below the stated value by, for example, 10% increments above or below (higher or lower).

As used herein, the term "adeno-associated virus" or "AAV" refers to all adeno-associated viruses used in gene therapy methods, including derivatives, viral subtypes and naturally occurring and recombinant forms thereof. Various serotypes of AAV can be used as recombinant gene transfer viruses to transduce many different cell types. Non-limiting examples of AAV serotypes are AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5) , AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primates genus AAV, non-primate AAV and ovine AAV. In some embodiments, the AAV is AAV type 8 (AAV-8) or AAV type 9 (AAV-9).

The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. The genome contains three genes rep (replication), cap (capsid) and aap (assembly). These three genes generate at least nine gene products through the use of three promoters, alternative translation initiation sites and differential splicing. AAV vectors of the present disclosure can contain additional elements that act in cis or trans. In certain embodiments, the AAV vector comprising the vector genome also contains one or more inverted terminal repeat (ITR) sequences flanking the 5' or 3' ends of the donor sequences; elements, tissue-specific expression control elements, intron sequences, stuffer or filler polynucleotide sequences, expression control elements (e.g., promoters or enhancers) that allow the donor sequence to be transcribed; and/or a polyadenine sequence located 3' to the donor sequence. have

AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA to cells. AAV infection of cells in culture is generally noncytopathic, and natural infection of humans and other animals is asymptomatic and asymptomatic. In addition, AAV infects many different types of mammalian cells and has the potential to target many individual tissues in vivo. AAV also promotes weaker immune responses compared to other forms of gene transfer, and has been shown to be a useful tool for gene transfer, including persistent expression as a non-integrating vector in both dividing and arresting cells. It has additional advantages that make it a particularly attractive virus system. Also, AAV tolerates the conditions used to inactivate adenovirus (56° C.-65° C. for several hours), making cryopreservation of rAAV-based vaccines less important. In some implementations, AAVs of the present disclosure include any one or more of these features.

As used herein, the term "AAV vector" includes or is derived from components of an adeno-associated virus (AAV), which may be used in brain, heart, lung, skeletal muscle, liver, kidney, spleen or pancreas. It refers to any vector suitable for infecting mammalian cells, whether in vitro or in vivo, including human cells of any of a number of tissue types, such as . The term "AAV vector" may be used to refer to an AAV-type viral particle (or virion) containing at least a nucleic acid molecule encoding a protein of interest. In some embodiments, the AAV vector contains a polynucleotide sequence (i.e., a polynucleotide that is heterologous to AAV, such as an anti-FAM19A5 antibody) rather than an AAV origin, typically a sequence of interest for genetic modification of cells. A "recombinant AAV vector" refers to an AAV vector containing Generally, the heterologous polynucleotide is flanked by at least one and typically two AAV inverted terminal repeats (ITRs). The term AAV vector includes both AAV vector particles and AAV vector plasmids.

As used herein, the term "ITR" refers to inverted terminal repeats. Typically, ITRs are involved in parvoviral (eg, AAV) DNA replication and rescue or excision from prokaryotic plasmids (Daya, S., et al, Clin Microbiol Rev 21(4):583-593 ( 2008)). ITRs are generally considered to be the minimal sequences required for AAV proviral integration and packaging of AAV DNA into virions. These elements are therefore essential for efficient propagation of the parvoviral genome.

As used herein, the term "serotype" refers to a subdivision of AAV that can be identified by serological or DNA sequence analysis methods and that is distinguishable by its antigenic properties. Also, when the term "isolate" is used in reference to AAV, it means a particular AAV serotype obtained from a given source. A person skilled in the art will recognize the difference between "isolated AAV", which refers to the relative purity of an AAV sample, and "AAV isolate", which refers to a clonally derived preparation of a particular AAV serotype, from the context in which the terms are used. will be easily recognized.

The terms "non-capsid" or "non-capsid" (or variations thereof) vector or nucleic acid molecule refer to a non-capsid vector construct. In some embodiments, the non-capsidated vector or nucleic acid molecule does not include, for example, a sequence encoding an AAV Rep protein.

In some embodiments, the AAV disclosed herein use a helper virus to replicate. As used herein, "helper virus" for AAV refers to a virus that enables AAV (eg, wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and fox viruses such as vaccinia. Although adenovirus type 5 of subfamily C is the most commonly used, said adenoviruses comprise a number of different subgroups. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Examples of herpetic viruses include giant cell virus (CMV) and pseudorabies virus (PRV), which are also obtained from depositories such as the ATCC, as well as herpes simplex virus (HSV) and Epstein-Barr virus (EBV). .

In some embodiments, the AAVs of the present disclosure have distinct tissue targeting capabilities (eg, tissue tropisms). The type further exhibits increased transduction or tropism compared to the non-variant parent capsid polypeptide.In some embodiments, the human stem cell type is an embryonic stem cell, an adult tissue stem cell ( i.e., somatic stem cells), bone marrow, progenitor cells, induced pluripotent stem cells, and reprogrammed stem cells.In some embodiments, adult stem cells are Organoid stem cells (i.e., stem cells derived from any organ or organ system of interest in the body) In some embodiments, the target tissue of AAV is the gonad, diaphragm, heart, stomach, liver, spleen, pancreas or kidney In some embodiments, the target body organ of the AAV is skin, hair, nails, sensory receptors, sweat glands, oil glands, bones, muscles, brain, Spinal cord, nerves, pituitary gland, pineal gland, hypothalamus, thyroid gland, parathyroid gland, thymus, adrenal gland, pancreas (islet tissue), heart, blood vessels, lymph nodes, lymph vessels, thymus, spleen, tonsils, nose, pharynx , larynx, respiratory tract, bronchi, lungs, mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, anal canal, teeth, salivary glands, tongue, liver, gallbladder, pancreas, appendix, kidneys, ureter, bladder, urethra, urethra Including, but not limited to, rounds, vas deferens, urethra, prostate, penis, scrotum, ovaries, uterus, fallopian tubes (trumpet tubes), vagina, vulva and mammary glands (breasts) Organs of the body Systems include, but are not limited to, the integumentary, skeletal, muscular, nervous, endocrine, cardiovascular, lymphatic, respiratory, digestive, urinary and reproductive systems. In some embodiments, transduction and/or tropism is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% , about 50%, about 55%, about 60%, 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. In some embodiments, transduction and/or tropism is increased by about 5% to about 80%, about 10% to about 70%, about 20% to about 60%, or about 30% to about 60%.

Although the terms "tropism" and "transduction" are interrelated, there are differences. As used herein, the term "tropism" refers to the ability of an AAV vector or virion to infect one or more specific cell types, although the vector may transduce cells into one or more specific cell types. It also includes whether it is functional; i.e., tropism refers to the preferential introduction of an AAV vector or virion into a particular cell or tissue type and/or a preference with a cell surface that facilitates entry into a particular cell or tissue type. expression (e.g., transcription and optionally translation) of sequences carried by the AAV vector or virion, optionally and preferably in a cell, e.g., in the case of recombinant viruses, expression of heterologous nucleotide sequences. means As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more specific cell types; It refers to the transfer of genetic material contained within a vector or virion into a cell to obtain expression from the vector genome. In some, but not all cases, transduction and tropism may be correlated.

As used herein, the term "multiplicity of infection" or "MOI" refers to the ratio of integrating vector to host cells used during transduction or transduction of host cells. For example, if 1,000,000 vectors are used to transduce 100,000 host cells, the multiplicity of infection is 10. The use of this term is not limited to transduction, but instead includes introduction of the vector into the host by methods such as lipofection, microinjection, calcium phosphate precipitation and electroporation.

The term "neuropathic pain" means pain due to injury, damage and/or improper function affecting any level of the central nervous system (CNS) and/or peripheral nervous system. The term "neuropathic pain" includes some and all types of neuropathic pain, regardless of the cause and some or all of the symptoms of neuropathic pain.

Neuropathic pain includes central neuropathic pain and peripheral neuropathic pain. As used herein, the term "central neuropathic pain" refers to pain resulting from a disorder, congenital defect, or central nervous system (ie, brain or spinal cord) injury. As used herein, the term "peripheral neuropathic pain" refers to pain resulting from injury or infection of peripheral sensory nerves.

Symptoms of neuropathic pain include persistent/chronic pain, spontaneous pain, as well as allogeneic pain (e.g., pain response to normally nonpainful stimuli), hyperalgesia (e.g., commonly known as simple pinprick pain). hyperesthesia (e.g., excessive physical sensitivity to stimuli, especially cutaneous stimuli) or hyperalgesia (e.g., brief inconvenience leading to severe pain over a long period of time) case) can be included. In some embodiments, symptoms may persist for a long time and may persist after the primary cause, if any, is resolved. Merck Manuals, Neuropathic Pain, merck manuals. com/professional/neurologic-disorders/pain/neuropathic-pain; N. and Meyer R. A. Neuron 52(1):77-92 (2006).

In some embodiments, the types of neuropathic pain are (1) neuralgia, (2) deafferentation pain syndrome, (3) complex site pain syndrome (CRPS), and (4) neuropathic pain syndrome. Pathology (central or peripheral) can be included.

In some embodiments, said neuropathic pain is generally neuralgia, which refers to pain that radiates along the pathways of one or more specific nerves (e.g., cranial nerves) without any overt pathological changes in nerve structure. be. Neuralgia includes trigeminal neuralgia (TN), atypical trigeminal neuralgia (ATN), laryngeal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia (induced by herpes zoster or herpes), peripheral nerve injury pain, sciatica, low back pain and atypical Including but not limited to facial pain. Chemical irritation, chronic kidney disease, diabetes, inflammation, trauma (including surgery), nerve compression by nearby structures (e.g. tumors), certain drugs (e.g. cisplatin, paclitaxel or vincristine), porphyrias (blood disorders), and Any infection (eg, herpes zoster, HIV/AIDS, Lyme disease or syphilis) can lead to neuralgia.

In some embodiments, the neuropathic pain may result from a loss of sensory input from a part of the body (e.g., caused by nerve blockade of peripheral sensory fibers or central nervous system). Pain syndrome. Deafferentation pain syndromes include, but are not limited to, brain or spinal cord injury, post-stroke pain, phantom pain, leg paralysis, brachial plexus avulsion injury and lumbar lumbar nerve root lesions. do not have.

In some embodiments, the neuropathic pain is "complex site pain syndrome" (CRPS), a chronic pain condition that most commonly affects the arms or legs. In some embodiments, the CRPS develops after injury, surgery, stroke or heart attack. In certain embodiments, the CRPS is CRPS type I (CRPS-I) (also known as reflex sympathetic neuropathy syndrome). Individuals with undiagnosed nerve damage are often classified as having CRPS-1. In another embodiment, the CRPS is Type II CRPS associated with confirmed nerve injury (CRPS-II) (also known as burning pain).

In some embodiments, the neuropathic pain is a neuropathy, which refers to pain resulting from functional or pathological alterations (eg, disease or injury) of nerves. Neuropathies can usually be characterized clinically by sensory or motor neuron abnormalities. In certain embodiments, the neuropathy is a central neuropathy (eg, functional or pathological changes in the central nervous system). In other embodiments, the neuropathy is peripheral neuropathy (eg, functional or pathological changes in one or more peripheral nerves, including motor nerves, sensory nerves, autonomic nerves, or combinations thereof). In some embodiments, the peripheral neuropathy involves functional or pathological changes to a single nerve or nerve group (ie, mononeuropathy). In some embodiments, the peripheral neuropathy is accompanied by functional or pathological changes affecting various nerves (locally or systemically) (ie, polyneuropathy). In some embodiments, the peripheral neuropathy affects both sides of the body substantially identically (ie, symmetric polyneuropathy). In some embodiments, the peripheral neuropathy affects disparate areas of the body (eg, mononeuritis multifocal, mononeuropathy multifocal, or mononeuropathy multifocal).

As used herein, "mononeuropathy" is a peripheral neuropathy involving loss of movement or sensation to areas caused by injury or destruction of a single peripheral nerve or nerve group. Mononeurosis is most often caused by local site injury or trauma, which, for example, causes prolonged pressure/compression on a single nerve. However, it can cause specific systemic disorders (eg, mononeuritis multiplex) and mononeurosis. In some embodiments, injury or trauma to the local site may cause partial destruction of the nerve's myelin sheath (integument) or nerve cells (axon) to slow or prevent the conduction of stimulation through the nerve. can. In some embodiments, the mononeuropathy can affect any part of the body. Examples of mononeuropathic pain are sciatic nerve dysfunction, common nasal nerve dysfunction, lumbar nerve dysfunction, spinal nerve dysfunction, cranial neuropathy VI, cranial neuropathy VII, cranial single neuropathy Neuropathy III (compression type), cranial mononeuropathy III (diabetic type), axillary nerve dysfunction, wrist tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, facial nerve palsy (Bell's palsy), thoracic exit Syndrome, Wrist Tunnel Syndrome and Sixth (Abducens) Nerve Palsy, including but not limited to. Finerup NB et al. , Pain 157(8): 1599-1606 (2016); ninds. nih. gov/disorders/peripheral neuropathy/detailj3 peripheral neuropathy. National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy Fact Sheet, available from www.htm. In some embodiments, the mononeuropathic pain is sciatica.

As used herein, "polyneuropathy" is a peripheral neuropathy involving loss of movement or sensation to areas caused by damage or destruction of multiple peripheral nerves. Polyneuropathic pain includes post-epileptic syndrome, post-mastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiency, chemotherapy-induced pain, 2' , 3′-dideoxycytidine (ddC) treatment, Guillain-Barré syndrome or Fabry disease. Finnerup N.W. B. et al. , Pain 157(8): 1599-1606 (2016); National Institute of Neurological Disorders and Stroke, ninds. nih. gov/disorders/peripheral neuropathy/detail3 peripheral neuropathy. Peripheral Neuropathy Fact Sheet available from htm. In some embodiments, the polyneuropathy is diabetic peripheral neuropathy. In certain embodiments, the diabetic peripheral neuropathy is caused by high blood glucose levels (blood sugar) and/or high levels of fats (e.g., triglycerides) in the blood of a diabetic subject, which can affect the patient's peripheral nerves. damage the

In some embodiments, the peripheral neuropathies disclosed herein can be classified based on the part of the nerve cell that is damaged or affected (eg, axon, myelin sheath or cell body). In some embodiments, the peripheral neuropathy is distal axonopathy due to axonal metabolic or toxic abnormalities. In some embodiments, the metabolic disorders include diabetes, renal failure, deficiency syndromes such as malnutrition, and alcoholism. In some embodiments, the metabolic disorder is diabetes and the distal axonopathy is diabetic neuropathy.

In some embodiments, the peripheral neuropathy is a myelination due to a primary attack on myelin or myelinated Schwann cells causing acute failure of conduction. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, also known as Guillain-Barré syndrome), but other causes include chronic inflammatory demyelinating syndrome (CIDP). ), inherited metabolic disorders (eg, leukodystrophy) or toxins.

In some embodiments, the peripheral neuropathy is a neuropathy due to destruction of peripheral nervous system (PNS) neurons. In some embodiments, the neuropathy is caused by motor neuron disease, sensory neuropathy (eg, herpes zoster), toxins, or autonomic dysfunction. In some embodiments, the neuropathy is induced by a neurotoxin, such as the chemotherapeutic agent vincristine.

Neuropathic pain can be caused by or associated with a variety of causes (e.g., physical injury (e.g., trauma or repetitive stress), disease or disorder, exposure to toxic substances, or combinations thereof). obtain. In some embodiments, the neuropathic pain is, for example, nerve compression injury (e.g., nerve crush, nerve outgrowth, nerve entrapment or incomplete neurotomy); spinal cord injury (e.g., hemisection of the spinal cord); peripheral Caused by injury or damage to nerves (e.g., motor, sensory, or autonomic nerves, or combinations thereof), limb amputation; contusion; inflammation (e.g., spinal cord inflammation); or traumatic injury or injury such as surgical procedures or related to it. In some embodiments, the neuropathic pain results from repetitive stress, including, for example, repetitive, inconvenient, and/or forceful activities that require movement of specific groups of joints for extended periods of time. or related to this. The resulting irritation, without being bound by any one theory, can cause ligaments, tendons and muscles to become inflamed and swollen, constricting narrow passageways for nerves (e.g. elbow or elbow). spinal neuropathy and wrist tunnel syndrome, which are neuropathies of trapped or compressed nerves in the wrist). In some embodiments, the neuropathic pain is associated with, for example, ischemic episodes (e.g., stroke or heart attack), multiple sclerosis, metabolic and/or endocrine diseases or disorders (e.g., diabetes, metabolic diseases and acromegaly, a condition induced by overproduction of growth hormone and characterized by abnormal enlargement of parts of the skeleton, including joints that cause nerve entrapment and pain); nerve tissue damage (e.g., vasculitis; vascular inflammation), autoimmune diseases (e.g., acute inflammatory demyelinating neuropathy, also known as Sjögren's syndrome, lupus, rheumatoid arthritis, and Guillain-Barré syndrome), causing a decrease in oxygen supply to peripheral nerves Diseases, renal disorders, cancers or tumors (e.g., angiogenic tumors, neuromas, paraneoplastic syndromes, toxicity of chemotherapeutic agents and radiation during cancer treatment, infections (e.g., herpes varicella (shingles), Epstein Infection by viruses such as Barr virus, West Nile virus, giant cell virus and herpes simplex virus, acquired immune deficiency syndrome (AIDS) or bacteria such as Lyme disease, diphtheria and leprosy), inflammatory disorders, peripheral neuropathy ( neuroma), congenital or de novo genetic disorders (e.g. Charcot-Marie-Tooth Disorder), severe weakness and loss of leg and foot muscles, abnormal gait , loss of tendon reflexes, leg paralysis), mononeuropathy or polyneuropathy.In some embodiments, the neuropathic pain is , caused by or associated with an infectious source (e.g., tick-borne infection, herpes varicella, Epstein-Barr virus, West Nile virus, giant cell virus, herpes simplex virus, AIDS). , said neuropathic pain may be caused by exposure to toxic substances including, for example, drugs, alcohol, heavy metals (e.g. lead, arsenic, mercury), industrial substances (e.g. solvents, fumes from adhesives) or nitrous oxide caused by or related to.

“Neuropathic pain associated with a disease or disorder” can be associated with a disease or disorder (e.g., those disclosed herein) or associated with a disease or disorder (e.g., those disclosed herein). Refers to neuropathic pain caused by or resulting from

The terms "treat", "treating" and "treatment" as used herein refer to the progression, development, severity of disease-related syndromes, complications, symptoms or biochemical signs. or any type of intervention or process performed on a subject or administering an active agent to a subject for the purpose of reversing, mitigating, ameliorating, inhibiting or delaying or preventing recurrence. Treatment may be administered to subjects with disease or subjects without disease (eg, prophylactically).

As used herein, “administering” refers to physically introducing a therapeutic agent or composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of ordinary skill in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the term "parenteral administration" generally refers to modes of administration other than enteral and topical administration by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal , intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intrablood, transtracheal, subcutaneous, subepidermal, intravitreal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and injection as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered by parenteral routes, such as topical, epidermal, or mucosal routes of administration, such as intranasal, oral, intravaginal, rectal, sublingual, or topical. Administration can also occur, for example, once, multiple times and/or over one or more extended periods of time.

As used herein, the term "therapeutically effective amount" refers to an amount that "treats" a disease or disorder in a subject or reduces the risk, potential, likelihood or occurrence of a disease or disorder (e.g., neuropathic pain). refers to the amount of drug, alone or in combination with other therapeutic agents, that is effective for A "therapeutically effective amount" is an amount of a drug or therapeutic agent that provides some improvement or benefit in a subject having or at risk of having a disease or disorder (e.g., neuropathic pain as disclosed herein). including. A "therapeutically effective amount" thereby reduces the risk, potential, likelihood or occurrence of a disease or disorder, or provides partial alleviation, alleviation, or at least one indication (e.g., neuropathic pain). ) and/or reduces at least one clinical symptom of the disease or disorder.

The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, eg non-mammals such as mammals and non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles and the like.

The term "family with sequence similarity 19, member A5" or "FAM19A5" belongs to the highly homologous TAFA family of 5 proteins (also known as the FAM19 family), which are predominantly expressed in the brain and spinal cord. refers to protein. FAM19A5 is also known as TAFA5 or chemokine-like protein TAFA-5.

The gene encoding FAM19A5 in humans is located on chromosome 22. There are multiple human FAM19A5 (UniProt: Q7Z5A7) isoforms that appear to be produced by alternative splicing: 132 amino acid isoform 1 (UniProt: Q7Z5A7-1), 125 and isoform 3 (UniProt: Q7Z5A7-3) consisting of 53 amino acids. The human FAM19A5 protein appears to exist in membrane-bound and soluble (secreted) forms. Isoform 1 appears to be a membrane with a single transmembrane region. Tang T. Y. et al. , Genomics 83(4):727-34 (2004) as a secreted protein (soluble) contains a signal peptide at amino acid positions 1-25. Isoform 1 appears to be a membrane protein. Following are the amino acid sequences of the three known human FAM19A5 isoforms.

(I) Isoform 1 (UniProt: Q7Z5A7-1, transmembrane protein): This isoform was selected as the standard sequence.

MAPSPRTGSR QDATALPSMS STFWAFMILA SLLIAYCSQL AAGTCEIVTL DRDSSQPRRT IARQTARCAC RKGQIAGTTR ARPACVDARI IKTKQWCDML PCLEGEGCDL LINRSGWTCT QPGGRIKTTT VS (SEQ ID NO: 1)
(II) Isoform 2 (UniProt: Q7Z5A7-2, soluble protein):
MQLLKALWAL AGAALCCFLV LVIHAQFLKE GQLAAGTCEI VTLDRDSSQP RRTIARQTAR CACRKGQIAG TTRARPACVD ARIIKTKQWC DMLPCLEGEG CDLLINRSGW TCTQPGGRIK TTTVS (SEQ ID NO: 2)
(III) Isoform 3 (UniProt: Q7Z5A7-3):
MYHHREWPAR IIKTKQWCDM LPCLEGEGCD LLINRSGWTC TQPGGRIKTT TVS (SEQ ID NO: 3)
The term "FAM19A5" includes any variant or isoform of FAM19A5 that is naturally expressed by the cell. Thus, the antibodies described herein may cross-react with different isoforms within the same species (e.g., different isoforms of human FAM19A5) or cross-react with FAM19A5 of species other than human (e.g., murine FAM19A5). can react. Alternatively, the antibody may be specific for human FAM19A5 and not show any cross-reactivity with different species. FAM19A5 or any variants and isoforms thereof can be isolated from cells or tissues that naturally express them, or produced recombinantly. A polynucleotide encoding human FAM19A5 has GenBank accession number BC039396, and a sequence as follows:

The term "antagonist against FAM19A5 protein" refers to all antagonists that suppress the expression of FAM19A5 protein. Such antagonists can be peptides, nucleic acids or chemical compounds. More specifically, the antagonist can be an antisense oligonucleotide targeting FAM19A5, siRNA, shRNA, miRNA, dsRNA, aptamer, PNA (peptide nucleic acid) or a vector comprising the same. In some embodiments, the antagonist can be an antibody or antigen-binding portion thereof that specifically binds to the FAM19A5 protein.

The terms "antibody" and "antibodies" are terms of the art and can be used interchangeably herein to refer to a molecule with an antigen-binding site that specifically binds to an antigen. . The term as used herein includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof. In some embodiments, "antibody" refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. In other embodiments, "antibody" refers to a single chain antibody comprising a single variable domain, eg, a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain in a particular naturally occurring antibody consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL.

The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each of VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. . The variable regions of the heavy and light chains contain binding domains that interact with antigen. The constant regions of the antibodies are capable of mediating binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. .

The term "Kabat numbering" and similar terms are art-recognized and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. point to In certain aspects, the CDRs of an antibody can be determined according to the Kabat numbering system (eg, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, an antibody heavy chain intramolecular CDR typically comprises amino acid positions 31-35 (CDR1) (optionally including one or two additional amino acids after position 35 (Kabat (referred to as 35A and 35B in the numbering system.), at amino acid positions 50-65 (CDR2) and at amino acid positions 95-102 (CDR3).Using the Kabat numbering system, the antibody light chain intramolecular CDRs are: Typically at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2) and amino acid positions 89-97 (CDR3) In certain embodiments, the antibodies described herein. The CDRs of are determined by the Kabat numbering system.

The phrases "same amino acid position numbering as in Kabat", "Kabat position" and grammatical variations thereof are defined in Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) for the heavy or light chain variable domains of antibody compilations. Using this numbering system, the actual linear amino acid sequence may contain additional amino acids corresponding to truncations or insertions into the FW or CDRs of the variable domain. For example, the heavy chain variable domain has a single amino acid inserted after residue 52 of H2 (residue 52a according to Kabat) and a residue inserted after residue 82 of the heavy chain FW (e.g. , according to Kabat, residues 82a, 82b and 82c, etc.). See Table 1B.

The Kabat numbering of residues for a given antibody can be determined by alignment in regions of homology of the antibody's sequence with a "standard" Kabat numbering sequence. Instead, Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loops vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system inserts H35A and H35B). without 35A or 35B, the loop ends at 32; with only 35A, the loop ends at 33; with both 35A and 35B, the loop ends at 34). The AbM hypervariable regions represent the negotiation between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, M.; P. et al. , Dev. Comp. Immunol. 27:55-77 (2003). The IMGT numbering system is based on alignments, structural data and hypervariable loop characterizations of over 5,000 sequences and facilitates comparison of variable and CDR regions for all species. According to the IMGT numbering scheme, VH-CDR1 is in positions 26-35, VH-CDR2 is in positions 51-57, VH-CDR3 is in positions 93-102, and VL- CDR1 is at positions 27-32, VL-CDR2 is at positions 50-52, and VL-CDR3 is at positions 89-97.

For all heavy chain constant region amino acid positions discussed in this disclosure, the numbering is based on that of Edelman et al. , 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85) according to the EU index first described. Edelman et al. The EU index of also Kabat et al. , 1991, Sequences of Proteins of Immunological Interest, 5th Ed. , United States Public Health Service, National Institutes of Health, Bethesda. Hereby the phrases "EU index as presented in Kabat" or "EU index as presented in Kabat" and "Position according to the EU index as presented in Kabat..." and their grammatical variants are derived from Edelman et al. refers to the residue numbering system based on the human IgG1EU antibody of .

The numbering system used for the variable domain (both heavy and light chain) and light chain constant region amino acid sequences is that provided in Kabat 1991.

An antibody can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any type (e.g., IgD, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subtype. (eg, IgG1, IgG2, IgG3 and IgG4 in humans; and IgG1, IgG2a, IgG2b and IgG3 in mice). Immunoglobulins, such as IgG1, exist in a variety of allotypes that differ from each other in at most a few amino acids. The antibodies disclosed herein can be of any of the commonly known isotypes, types, subtypes or allogeneic types. In certain embodiments, the antibodies disclosed herein are IgG1, IgG2, IgG3, or IgG4 subtypes or hybrids of any of these. In certain embodiments, the antibody has a human IgG1 subtype or a human IgG2 or human IgG4 subtype.

"Antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, fully synthetic antibodies; single chain antibodies; Multispecific antibodies (including bispecific antibodies); tetrameric antibodies comprising two heavy chain and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers antibody light chain-antibody heavy chain pair; intrabody; heteroconjugate antibody; monovalent antibody; single chain antibody; camelized antibody; Single domain antibodies (sdAbs), including anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and binding molecules consisting of a single monomeric variable antibody domain (e.g., VH domain or VL domain) fully capable of antigen binding (Harmen MM and Haard HJ Appl Microbiol Biotechnol. 77(1):13-22 (2007)).

As used herein, the terms "antigen-binding portion" and "antigen-binding fragment" of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human FAM19A5). point to Such "fragments" are, for example, from about 8 to about 1500 amino acids long, preferably from about 8 to about 745 amino acids long, preferably from about 8 to about 300, eg from about 8 to about 1500 amino acids long. It is about 200 amino acids or about 10 to about 50 or 100 amino acids long. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments included in the term "antigen-binding portion" of an antibody, e.g., an anti-FAM19A5 antibody disclosed herein are (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments joined by a disulfide junction at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) separate complementarity determining regions (CDRs) or (vii) a combination of two or more separate CDRs that can optionally be joined by a synthetic linker. Also, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they are combined using recombinant methods to pair the VL and VH regions to form a monovalent molecule. can be joined by a synthetic linker that can be made into a single protein chain (known as a single-chain Fv (scFv)); see, for example, Bird et al. , (1988) Science 242:423-426; and Huston et al. , (1988) Proc. Natl. Acad. Sci. See USA 85:5879-5883. Such single-chain antibodies are also included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

As used herein, the terms "variable region" and "variable domain" are interchangeable and universal in the art. Said variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino terminal 110-120 amino acids within said mature heavy chain. There are about 90-115 amino acids within the mature light chain that vary extensively in sequence between antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domains are called framework regions (FRs).

Without being bound by theory or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antigen-antibody interaction and specificity. In certain embodiments, said variable region is a human variable region. In certain embodiments, the variable regions comprise rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (eg, non-human primate) variable region. In certain embodiments, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

As used herein, the term “heavy chain (HC)” when used in reference to an antibody includes IgG subtypes, such as IgG1, IgG2, IgG3 and IgG4, based on the amino acid sequence of the constant domain, each Refers to any distinct type of generating IgA, IgD, IgE, IgG and IgM types of antibodies, such as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ) can be done.

As used herein, the term "light chain (LC)", when used in reference to an antibody, refers to any distinct type, e.g., kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. can point to Light chain amino acid sequences are well known in the art. In certain embodiments, said light chain is a human light chain.

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

As used herein, the terms "constant region" or "constant domain" are interchangeable and have their ordinary meaning in the art. Said constant domains are those of the antibody part, e.g. the light and/or heavy chains, which are not directly involved in the binding of the antibody to the antigen, but which may exhibit various effector functions such as interaction with Fc receptors. Carboxy-terminal portion. The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences than immunoglobulin variable domains.

"Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to Fc receptors located on various cells of the immune system (e.g., effector cells) or the classical complement system. The C-terminal region of the heavy chain of an antibody that contains binding to the first component (C1q) of the antibody and mediates binding of the immunoglobulin to host tissues or factors. The Fc region thereby comprises the constant region of an antibody, excluding the first constant region immunoglobulin domain (eg, CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains; IgM and IgE. The Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region includes the hinge between the immunoglobulin domains Cγ2 and Cγ3 and between Cγ1 and Cγ2. Although the boundaries of the Fc region of immunoglobulin heavy chains can vary, human IgG heavy chain Fc regions generally span from amino acid residues at positions C226 or P230 (or between those two amino acids). Numbering follows the EU index as in Kabat, although limited to those extended to the carboxy terminus of the chain. The CH2 domain of the human IgG Fc region is extended from about amino acid 231 to about amino acid 340, and the CH3 domain is located C-terminal to the Cm domain in the Fc region, i.e., about amino acids of IgG. It is extended from position 341 to about amino acid position 447. As used herein, an Fc region can be a native sequence Fc, including any allogeneic variant, or a variant Fc (eg, a non-naturally occurring Fc). Fc may also be associated with Fc-containing protein polypeptides such as "binding proteins comprising the Fc region", also referred to as "Fc fusion proteins" (e.g., antibodies or immunoadhesions), where this region is isolated. can be meant as

A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region as found in nature. A native sequence human Fc region includes a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes various allogeneic forms of Fc (e.g., Jefferis et al. (2009) mAbs 1:1; Vidarsson G. et al. Front Immunol. 5:520 (October 20, 2014 online). public)).

An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind IgG antibodies include the Fcγ family of receptors and allelic variants and otherwise spliced forms of these receptors. The Fcγ family consists of three activating receptors (murine FcγRI, FcγRIII and FcγRIV; human FcγRIA, FcγRIIA and FcγRIIIA) and one inhibitory receptor (FcγRIIB). Human IgG1 binds most human Fc receptors and elicits the strongest Fc effector function. Human IgG1 can be equated to murine IgG2a in relation to the type of activating Fc receptors to which it binds. Conversely, human IgG4 elicits minimal Fc effector function (see Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014)).

The constant region can be engineered, eg, by recombinant techniques, to remove one or more effector functions. "Effector function" refers to the interaction or biochemical response resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Exemplary "effector functions" include FcγR-mediated effector functions such as C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) and cell Including down-regulation of surface receptors (eg, B-cell receptor; BCR). Such effector function generally requires that the Fc region be combined with a binding domain (eg, an antibody variable domain). Thus, the term "Fc-function-free constant region" includes constant regions in which one or more effector functions mediated by the Fc region are reduced or absent.

Antibody effector functions can be reduced or avoided by different approaches. Antibody effector functions use antibody fragments lacking the Fc region (such as Fab, F(ab')2, single-chain Fv (scFv) or sdAbs consisting of monomeric VH or VL domains). can be reduced or avoided by In contrast, removal of sugars linked to specific residues in the Fc region reduces antibody effector function while retaining other valuable properties of the Fc region (e.g., long half-life and heterodimerization). A so-called aglycosylated antibody can be produced by letting it ferment. Aglycosylated antibodies are produced by enzymatically removing sugars, e.g., by deleting or altering the residues to which the sugars are attached, thereby injecting antibodies into cells cultured in the presence of a glycosylation inhibitor. production, or by expressing the antibody in a cell incapable of glycosylating protein (eg, a bacterial host cell). See, for example, US Patent Publication No. 20120100140. Another approach is to take advantage of the Fc regions of IgG subtypes that have reduced effector function, e.g., IgG2 and IgG4 antibodies have lower levels of Fc effector function compared to IgG1 and IgG3. characterized by Residues closest to the hinge region in the CH2 domain of the Fc portion are responsible for the antibody effector functions, but they are responsible for C1q (complement) and IgG-Fc receptors (FcγR) on the effector cells of the innate immune system. (Vidarsson G. et al. Front Immunol. 5:520 (published online Oct. 20, 2014)). Thus, an antibody with reduced or absent Fc effector function may be, for example, a chimeric Fc region comprising the CH2 domain of an IgG antibody of the IgG4 isotype and the CH3 domain of an IgG antibody of the IgG1 isotype, or a hinge region of IgG2 and an IgG4 (see, e.g., Lau C. et al. J. Immunol. 191:4769-4777 (2013)), or alter Fc effector function, e.g., reduce or eliminate Fc function. They can be produced by generating Fc regions with mutations. Such Fc regions with mutations are known in the art. For example, US Patent Publication No. 20120100140 and US and PCT applications cited therein and An et al. , mAbs 1:6, 572-579 (2009).

"Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region comprising the upper, middle and lower hinge joining the CH1 domain with the CH2 domain ( Roux et al., J. Immunol. 1998 161:4083). The hinge provides varying levels of flexibility between the binding and effector regions of the antibody and also provides the site for the intermolecular disulfide bond between the two heavy chain constant regions. As disclosed herein, the hinge begins at Glu216 and ends at Gly237 for all IgG isoforms (Roux et al., 1998 J Immunol 161:4083). The sequences of wild-type IgG1, IgG2, IgG3 and IgG4 hinges are known in the art. For example, Kabat EA et al. , (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.A.; S. Department of Health and Human Services, NIH Publication no. 91-3242; Vidarsson G.; et al. , Front Immunol. 5:520 (published online Oct. 20, 2014).

The term "CH1 domain" refers to the heavy chain constant region that joins the variable domains to the hinge at the heavy chain constant domain. As used herein, the CH1 domain starts at A118 and ends at V215. The term "CH1 domain" includes not only wild-type CH1 domains, but also naturally occurring variants thereof (eg, allogeneic forms). CH1 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild-type and allogeneic types) are known in the art (see, e.g., Kabat EA et al., (1991), supra and Vidarsson G. et al., supra). al., Front Immunol., 5:520, published online October 20, 2014. Exemplary CH1 domains include, for example, US Patent Publication No. 20120100140 and US patents, publications and PCTs cited therein. It includes a CH1 domain with mutations that alter the biological activity, eg, half-life, of the antibody described in the publication.

The term "CH2 domain" refers to the heavy chain constant region hinge-joining the CH3 domain at the heavy chain constant domain. As used herein, the CH2 domain begins at P238 and ends at K340. The term "CH2 domain" includes not only wild-type CH2 domains, but also naturally occurring variants thereof (eg, allogeneic forms). CH2 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild-type and allogeneic types) are known in the art (see, e.g., Kabat EA et al., (1991), supra and Vidarsson G. et al., supra). al., Front Immunol., 5:520, published online October 20, 2014. Exemplary CH2 domains are described, for example, in U.S. Patent Publication No. 20120100140 and U.S. patents, publications and PCTs cited therein. Include a CH2 domain with mutations that alter the biological activity of the antibody described in the publication, eg, half-life and/or reduced Fc effector function.

The term "CH3 domain" refers to the heavy chain constant region that is C-terminal to the CH2 domain in the heavy chain constant domain. As used herein, the CH3 domain begins at G341 and ends at K447. The term "CH3 domain" includes not only wild-type CH3 domains, but also naturally occurring variants thereof (eg, allogeneic forms). CH3 domain sequences of IgG1, IgG2, IgG3 and IgG4 (including wild-type and allogeneic types) are known in the art (see, e.g., Kabat EA et al., (1991), supra and Vidarsson G. et al., supra). al., Front Immunol., 5:520, published online October 20, 2014. Exemplary CH3 domains are described, for example, in U.S. Patent Publication No. 20120100140 and U.S. patents, publications and PCT publications cited therein. contains a CH3 domain with mutations that alter the biological activity, eg, half-life, of the antibodies described in .

As used herein, "isotype" refers to the antibody class (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

"Allogeneic" refers to naturally occurring variants within a particular isotype group that differ by a number of amino acids (see, eg, Jefferis et al., (2009) mAbs 1:1). The antibodies described herein can have any allogeneic form. Allogeneic forms of IgG1, IgG2, IgG3 and IgG4 are known in the art. For example, Kabat EA et al., supra. , (1991); et al. , Front Immunol. 5:520 (published online October 20, 2014); and Lefran MP, mAbs 1:4, 1-7 (2009).

The terms "antibody that recognizes an antigen" and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to FAM19A5 is (substantially no antibodies that specifically bind to the antigen of An isolated antibody that specifically binds an epitope of FAM19A5 may, however, have cross-reactivity to other FAM19A5 proteins of different species.

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site on a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise indicated, "binding affinity" as used herein means the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X for partner Y can generally be expressed by a dissociation constant (K _D ). Affinity can be measured and/or displayed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (K _D ) and equilibrium association constant (K _A ). The K _D is calculated from the quotient k _off /k _on and is expressed in molarity (M), and the K _A is calculated from the quotient k _on /k _off . k _on means eg the binding rate constant of an antibody to an antigen and k _off means eg the dissociation of an antibody to an antigen. k _on and k _off are determined by techniques known to those skilled in the art, such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)), BIAcore® or kinetic exclusion assays (KinExA®). can be

As used herein, the terms “specifically binding,” “specifically recognizing,” “specific binding,” “selectively binding,” and “selectively binding” are analogous terms in the context of antibodies. , refers to a molecule (eg, an antibody) that binds to an antigen (eg, an epitope or immune complex), binding being understood by those of skill in the art. For example, molecules that specifically bind to an antigen can be analyzed by, for example, immunoassays, BIAcore®, KinExA® 3000 instruments (Sapidyne Instruments, Boise, Id.) or other analytical methods known in the art. When determined, it is usually capable of binding other peptides or polypeptides with lower affinity. In certain embodiments, the molecule that specifically binds to an antigen has a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or more greater than _{the KA} when the molecule binds to another antigen. binds to antigens;

Antibodies typically specifically bind their cognate antigens with high affinity as reflected by a dissociation constant (K _D ) of 10 ⁻⁵ to 10 ⁻¹¹ M or less. Any K _D above about 10 ⁻⁴ M is generally considered to imply non-specific binding. As used herein, an antibody that "specifically binds" an antigen refers to an antibody that binds the antigen with high affinity and substantially the same antigen, for example, by BIACORE using the given antigen. 10 ⁻⁷ M or less, preferably 10 ⁻⁸ M or less, more preferably 10 ⁻⁹ M or less, most preferably as determined by immunoassay (e.g. ELISA) or surface plasma resonance (SPR) techniques on a ^TM 2000 instrument Although implied to have a K _D of 10 ⁻⁸ M to 10 ⁻¹⁰ M or less, this antibody does not bind irrelevant antigens with high affinity.

"Antigen" as used herein refers to any natural or synthetic immunogenic substance such as a protein, peptide or hapten. The antigen can be FAM19A5 or a fragment thereof.

As used herein, "epitope" is a term of art and refers to a localized region of an antigen capable of specific binding by an antibody. An epitope is, for example, a contiguous amino acid of a polypeptide (linear or contiguous epitope), or an epitope is, for example, a collection of two or more non-contiguous regions of a polypeptide or a polypeptide (conformal, non-linear, discontinuous or non-contiguous epitopes). Epitopes formed from contiguous amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically treated with denaturing solvents. lost at times. An epitope typically comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 amino acids in a unique spatial conformation. include. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, mapping overlapping or adjacent peptides (e.g. of FMAM19A5) to a given antibody (e.g. , immunoblot and immunoprecipitation analysis testing for reactivity with anti-FAM19A5 antibody). Methods of determining spatial conformation of epitopes include techniques in the art and described herein, such as x-ray crystallography, 2-dimensional nuclear magnetic resonance and HDX-MS (eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, GE Morris, Ed. (1996)).

In certain embodiments, the epitope to which the antibody binds is, for example, NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA analysis, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectroscopic analysis), array-based oligopeptide scanning analysis and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). For X-ray crystallography, determinization can be accomplished using any of the methods known in the art (eg, Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4):339-350; See McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques, distributed by X-PLOR (Yale University, 1992, Molecular Simulations, Inc.; see, eg, Meth Enzymol (1985) volumes 114 & 115, eds Wyck _off H. W. et al., see U.S. 2004/0014194) and BUSTER (Bricogne G. (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1):37-60; Bricogne G. (1997) Meth Enzymol 276A). Roversi P. et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those of skill in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, eg, Champe M.; et al. , (1995) J Biol Chem 270:1388-1394 and Cunningham B.M. C. & WellsJ. A. (1989) Science 244:1081-1085.

The term "epitope mapping" refers to the process of identifying the molecular determinants of antibody antigen recognition.

The term "binds to the same epitope" in the context of two or more antibodies means that the antibodies bind to the same segment of amino acid residues as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on FAM19A5" as an antibody described herein include epitope mapping methods, such as crystallography of antigen:antibody complexes, which provide atomic resolution of the epitope. x-ray analysis and hydrogen/deuterium exchange mass spectroscopy (HDX-MS). Another method is to monitor the binding of antibodies to antigen fragments or mutated variants of antigens, wherein loss of binding due to alteration of amino acid residues within the antigen sequence is often associated with epitope components. regarded as display. Computational combinatorial methods for epitope mapping are also available. These methods rely on the ability of the antibody of interest to affinity separate specific short peptides from combinatorial phage display peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind the same epitope.

An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) binding of said other antibody to a target. Whether or not two antibodies compete with each other for binding to the target, i.e., whether and to what extent one antibody inhibits the other antibody from binding to the target, can be determined using known competition experiments. can decide. In certain embodiments, one antibody competes with the other antibody for binding to the target and reduces this binding by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. Or inhibit up to 100%. The level of inhibition or competition may vary depending on whether the antibody is a "blocking antibody" (ie, a cold antibody that is first incubated with the target). Competitive analysis is described, for example, in E.M. d. Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb. prot 4277 or E. d. It can be performed as described in Chapter 11 of "Using Antibodies" by Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitopes or adjacent epitopes (as evidenced by steric hindrance, for example).

Other competitive binding assays include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., Methods in Enzymology 9:242 (1983)). solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); Direct biotin-avidin EIAs (see Cheung et al., Virology 176:546 (1990)); and direct labeling RIAs (see Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

The term "single-chain Fv" or "scFv" refers to a single-chain Fv ("variable fragment") antibody in which the heavy chain and single-chain variable domains of a traditional double-chain antibody are joined to form one chain. point to

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody in which two different heavy/light chain pairs have two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al. , J. Immunol. 148, 1547-1553 (1992).

As used herein, a "monoclonal antibody" is an antibody that exhibits a single binding specificity and affinity for a particular epitope, or all antibodies exhibit a single binding specificity and affinity for a particular epitope. It refers to the composition of an antibody that exhibits sex. Accordingly, the term "human monoclonal antibody" refers to an antibody or antibody composition displaying a single binding specificity and having variable and alternative constant regions derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is a transgenic non-human animal, e.g., a transgenic non-human animal, e.g. Produced by hybridomas containing B cells obtained from mice.

As used herein, the term "recombinant human antibody" refers to (a) an antibody that has been transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma produced therefrom, isolated from an animal (e.g., a mouse); (b) antibodies isolated from host cells that have been transformed to express the antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) ) produced, expressed, produced or isolated by recombinant means, such as antibodies produced, expressed, produced or isolated by any other means involving splicing the human immunoglobulin gene sequences to other DNA sequences; Includes all human antibodies. Such recombinant human antibodies use specific human germline immunoglobulin sequences encoded by germline genes, but include, for example, variable and constant regions that contain subsequent rearrangements and mutations that occur during antibody maturation. . As known in the art (see, eg, Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable regions are rearranged to form antibodies specific to foreign antigens. contains antigen-binding domains encoded by various genes. Besides rearrangements, the variable regions can be further modified by a number of single amino acid changes (called somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will change according to the additional response to antigen (ie, isotype change). Thus, rearranged and somatically mutated nucleic acid molecules that encode light and heavy immunoglobulin polypeptides in response to antigen may not have sequence identity to the original nucleic acid molecule, but instead , will be substantially identical or similar (ie, have at least 80% identity).

A "human antibody (HuMAb)" refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Alternatively, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein have amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by in vivo somatic cell mutation). can include However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids derived from human immunoglobulin. In some embodiments of humanized forms of antibodies, some, most, or all of the amino acids outside the CDR domains are replaced with human immunoglobulin amino acids, and amino acids inside one or more of the CDR regions are replaced with human immunoglobulin amino acids. Some, most or all remains unchanged. Minor additions, deletions, insertions, substitutions or variations of amino acids are permissible as long as they do not destroy the antibody's ability to bind to the specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

A “chimeric antibody” is an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. point to

As used herein, the term "cross-reactivity" refers to the ability of the antibodies disclosed herein to bind to different species of FAM19A5. For example, antibodies disclosed herein that bind human FAM19A5 can also bind FAM19A5 of other species (eg, murine FAM19A5). Cross-reactivity, as used herein, is defined by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA) or binding to cells that physiologically express FAM19A5. It can be measured by functionally interacting with said cell. Methods for determining cross-reactivity can be performed using standard binding assays as described herein, e.g., BIACORE ^™ Surface Plasma Resonance (SPR) analysis using a BIACORE ^™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden). , or including flow cell counting techniques.

When the term "naturally occurring" as disclosed herein is applied to an object, it refers to the fact that the object can be found from nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that is separable from a natural source and not intentionally modified by humans in a laboratory is naturally occurring.

A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, and there is no upper limit to the length of said chain. One or more amino acid residues within a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. A "protein" can include one or more polypeptides.

The term "nucleic acid molecule" as used in the present invention includes DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell containing nucleic acid that is not naturally occurring within the cell, and into which a recombinant expression vector has been introduced. could be. Such terms should be understood to refer not only to the particular subject cell, but also to the progeny of such cells. Such progeny are not actually identical to the parent cell, since certain modifications may occur in subsequent generations due to mutations or environmental influences, but the term "host cell" as used herein ” still falls within the scope of

As used herein, the term "linked" refers to the association of two or more molecules. Said linkage may be a covalent linkage or a non-covalent linkage. Said linkage can also be a genetic linkage (ie, fusion by recombination). Such linkages can be accomplished using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

<II. Adeno-associated virus (AAV) and vectors derived therefrom>
The AAV vectors disclosed herein comprise nucleic acid encoding a protein of interest, eg, a FAM19A5 antagonist, eg, an anti-FAM19A5 antibody. In some embodiments, the nucleic acids are also expression and, in some embodiments, sequences encoding, for example, promoters, enhancers, polyadenylation signals, internal liposome entry sites (IRES), protein transduction domains (PTDs), etc. It can contain one or more regulatory sequences that enable secretion of the protein of interest, such as. Thus, in some embodiments, the nucleic acid can include a promoter region operably linked to the coding sequence to induce or improve expression of the protein of interest in infected cells. Such promoters can be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, e.g., allowing efficient and stable production of proteins in infected tissues. can be Generally, the promoters used in the disclosed methods are functional in human cells, but the promoter can be homologous or heterologous to the encoded protein. Examples of regulated promoters include, but are not limited to, Tet on/off element-containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters and metallothionein promoters. Other promoters that can be used include promoters that are tissue specific for tissues such as kidney, spleen, pancreas, brain or spinal cord. Examples of ubiquitous promoters include, among others, viral promoters such as the CMV promoter, RSV promoter, SV40 promoter, and cellular promoters such as the PGK (phosphoglycerate kinase) promoter and the β-actin promoter.

In some embodiments of the AAV vectors disclosed herein, one or more feedback elements can be used to attenuate overexpression of the protein of interest. For example, some embodiments of the AAV vectors contain one or more siRNA sequences that target exogenous transcripts. In other embodiments, the AAV vector contains one or more additional promoters that can be recognized by inhibitory transcription factors. In various embodiments, the AAV vectors disclosed herein contain constructs capable of generating a homoeostatic feedback loop that can maintain the expression level of the protein of interest at physiological levels.

In some embodiments, the AAV vectors disclosed herein contain nucleic acid that includes a leader sequence (or signal peptide) that allows secretion of the encoded protein. In some embodiments, a form of therapy that can be secreted from transduced cells by fusing the transgene of interest to a sequence encoding a secretory signal peptide (usually located at the N-terminus of the secreted polypeptide). It can allow protein production. Non-limiting examples of such signal peptides include human beta globin, albumin, beta-glucuronidase, alkaline protease or fibronectin secretory signal peptides.

In some embodiments, the mRNA expressed from the AAV vectors of the present disclosure contains miRNA binding sites for miRNAs preferentially expressed from non-CNS tissues. In certain embodiments, said miRNA binding site is a binding site for miR-122. In certain embodiments, said miRNA binding site is a binding site for miR-1. In some embodiments, the mRNA expressed from the CNS-associated gene does not contain miRNA binding sites for miRNAs preferentially expressed from CNS tissues.

AAV, which belongs to the Parvoviridae family, is a small unenveloped icosahedral virus with a single-stranded linear DNA genome of 4.7 kilobases (kb) to 6 kb. Gonqalves, M.; , Virol J2:43 (2005). The family is divided into two subfamilies: the Parvoirineae, which infect vertebrates, and the Densoirineae, which infect insects. Viruses belonging to the parvoirhinae subfamily, referred to herein as parvoviruses, include the genus Densovirus. As can be inferred from their genus name, viruses belonging to the Dependoviruses are in that they generally require co-infection with a helper virus, such as adenovirus or herpes virus, for productive infection in cell culture. is unique in Dependoviruses commonly infect AAV and other warm-blooded animals that infect humans (e.g. serotypes 1, 2, 3A, 3B, 4, 5 and 6) or primates (e.g. serotypes 1 and 4). related viruses (eg, bovine, canine, equine, and ovine adeno-associated viruses). Additional information on parvoviruses and other viruses belonging to the parvoviridae family can be found in Kenneth I.; Bems, "Parvoviridae: The Viruses and Their Replication", Chapter 69 in Fields Virology (3d Ed. 1996).

<Replication, capsid and assembly AAV genes>
The single-stranded genome of AAV contains three genes, rep (replication), cap (capsid) and aap (assembly). These three genes generate at least nine gene products through the use of three promoters, alternate translation initiation sites and differential splicing.

The rep gene encodes four proteins (Rep78, Rep68, Rep52 and Rep40) required for viral genome replication and packaging. The cap gene expression generates viral capsid proteins (VP1; VP2; VP3) that not only protect the viral genome, but also form an outer capsid shell that is actively involved in cell binding and internalization. The viral coat is presumed to consist of 60 proteins arranged in an icosahedral structure. The aap gene encodes an assembly-activating protein (AAP) in an alternate reading frame that overlaps with the cap gene. Such nuclear proteins are thought to provide scaffolding functions for capsid assembly and play a role in the nuclear localization of VP proteins in some AAV serotypes.

In some embodiments, the AAV vectors disclosed herein are recombinant AAV, lacking any one or more of the rep, cap and aap genes. In some embodiments, the rAAV is modified such that any one or more of the rep, cap and aap genes is mutated, and the expression of any one or more of the AAV genes is modified. In some embodiments, any one or more of the rep, cap, or aap genes are naturally occurring, e.g., the rep, cap, or app genes are all of the parvoviral rep, cap, or aap genes, or Including some. In some embodiments, any one or more of the rep, cap or aap genes comprise synthetic sequences.

It is understood that the particular AAV genomes described herein may have different AAV genomes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 genomes). obtain. Hereby, the specification discloses AAV vectors comprising any possible permutations of rep, cap or aap.

<Inverted terminal repeat>
A particular aspect of the present disclosure relates to nucleic acid molecules that include a first ITR, such as the 5'ITR, and a second ITR, such as the 3'ITR. In some embodiments, the ITR is naturally occurring, eg, the ITR comprises all or part of a parvoviral ITR. In some embodiments, the ITRs comprise synthetic sequences.

In some embodiments, the ITRs comprise ITRs of the AAV genome. In some embodiments, the ITR is an AAV genomic ITR selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. In certain embodiments, the ITRs are ITRs of the AAV8 or AAV9 genome. In other embodiments, the ITRs are synthetic sequences genetically engineered to include ITRs from any one or more of the AAV genomes at the 5' and 3' ends. In some embodiments, the ITRs are derived from the same genome, eg, from the same viral genome, or from different genomes, eg, from two or more different AAV genomes. In certain embodiments, the ITRs are derived from the same AAV genome. In other embodiments, the two ITRs present in the nucleic acid molecules of the present disclosure can be the same, particularly AAV2 ITRs. In some implementations, the first ITR and the second ITR are the same.

In some embodiments, the ITRs are synthetic sequences that have been engineered to include ITRs at the 5' and 3' ends that do not originate from the AAV genome. In some embodiments, the ITR sequences comprise one or more palindromic sequences. The palindromic sequences of the ITRs disclosed herein include natural palindrome sequences (ie, sequences found in nature), synthetic sequences such as analogous palindrome sequences (ie, sequences not found in nature), and combinations thereof. or variations, including but not limited to. A "similar palindromic sequence" is less than 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% relative to a sequence within a native AAV palindrome that forms secondary structure. are palindromic DNA sequences that share less than 80% identity or contain imperfect palindrome sequences that have no nucleic acid sequence identity. Said natural palindrome sequence can be obtained or derived from any genome disclosed herein. The synthetic palindrome can be based on any genome disclosed herein. The palindrome may be continuous or interrupted. In some embodiments, the palindromes are discontinuous, including insertions of secondary sequences. In some embodiments, the second sequence is a promoter, an enhancer, an integration site for integrase (e.g., a site for Cre or Flp recombinase), an open reading frame for the gene product, or a combination thereof. including.

In some embodiments, the ITRs form hairpin loop structures. In one embodiment, the first ITRs form a hairpin structure. In another embodiment, the second ITR forms a hairpin structure. In yet another embodiment, both the first ITR and the second ITR form a hairpin structure.

In some embodiments, the ITRs of the nucleic acid molecules described herein can be transcriptionally activated ITRs. A transcriptionally activated ITR can comprise all or part of a transcriptionally activated wild-type ITR by including at least one transcriptionally activated element. Various types of transcriptional activation elements can be suitably used in this connection. In some embodiments, the transcriptional activation element is a constitutive transcriptional activation element. Constitutive transcriptionally active elements provide sustainable levels of gene transcription and are preferred when sustained expression of the transgene is required. In another embodiment, said transcriptional activation element is an inducible transcriptional activation element. Inducible transcriptional activation elements generally exhibit low activity in the absence of an inducer (or inducing conditions) and are upregulated in the presence of an inducer (or switched to inducing conditions). Inducible transcriptional activation elements may be preferred when expression is required only at specific times or locations, or when expression levels need to be titrated using an inducing agent. Transcriptional activation elements may also be tissue-specific, ie exhibit activity only in particular tissues or cell types.

Transcriptional activation elements can be incorporated into ITRs in a variety of ways. In some embodiments, the transcriptional activation element is 5' spliced to any portion of the ITR, or 3' splinted to any portion of the ITR. In other embodiments, the transcriptionally active element of a transcriptionally activated ITR is located between two ITR sequences. If the transcriptionally active element comprises two or more elements that are to be separated, these elements may alternate with portions of the ITR. In some embodiments, the ITR hairpin structure is deleted and replaced by an inverted repeat of the transcriptional element. Such latter sequences will produce a hairpin that mimics the missing portion in structure. There may be multiple tandem transcriptional activation elements in a transcriptionally activated ITR, which may be contiguous or remote. Also, a protein binding site (eg, a Rep binding site) can be introduced into the transcriptional activation element of a transcriptionally activated ITR. A transcriptional activation element can comprise any sequence that allows for the regulated transcription of DNA by an RNA polymerase to form RNA, and can include, for example, the transcriptional activation elements defined below.

Transcriptionally activated ITRs provide both transcriptional activation and ITR functions to a nucleic acid molecule with a relatively restricted nucleotide sequence length that effectively maximizes the length of the transgene that can be transported and expressed from the nucleic acid molecule. . Incorporating transcriptionally active elements into ITRs can be accomplished in a variety of ways. A comparison of the sequence requirements of ITR sequences and transcriptional activation elements can provide insight into how elements within ITRs are encoded. For example, transcriptional activity can be added to an ITR by introducing specific changes within the ITR sequence that duplicate the functional elements of the transcriptional activation element. Numerous techniques exist in the art for efficiently adding, deleting and/or altering specific nucleotide sequences at specific sites (see, eg, Deng and Nickoloff (1992) Anal. Biochem. 200:81-88). reference). Another method of generating transcriptionally activated ITRs involves introducing restriction sites at desired positions in the ITR. Also, multiple transcriptional activation elements can be incorporated into transcriptionally activated ITRs using methods known in the art.

By way of illustration, transcriptionally activated ITRs include the TATA box, GC box, CCAAT box, Spl site, Inr region, CRE (cAMP regulatory element) site, ATF-1/CRE site, ARBb box, APBa box, CArG box, It may be generated by including one or more transcriptionally active elements such as a CCAC box or any other element involved in transcription known in the art.

<FAM19A5 antagonist>
AAV vectors of the present disclosure comprise nucleic acids encoding one or more antagonists of the FAM19A5 protein. In some embodiments, the FAM19A5 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to the FAM19A5 protein. In some embodiments, the anti-FAM19A5 antibody comprises Fab, Fab', F(ab')2, Fv or single chain Fv (scFv). In certain embodiments, said anti-FAM19A5 antibody is a scFv.

In some embodiments, anti-FAM19A5 antibodies (eg, anti-FAM19A5 scFv) expressed by AAV vectors of the disclosure are characterized by particular functional characteristics or properties. For example, the antibody specifically binds human FAM19A5, including soluble FAM19A5 and membrane-bound FAM19A5. In addition to specific binding to soluble and/or membrane-bound human FAM19A5, the antibodies described herein exhibit any one or more of the following functional properties:
(a) fibrosis reduction, reversal and/or prevention;
(b) reduced formation of excessive extracellular matrix (ECM);
(c) slowing tumor growth or progression;
(d) binds to soluble human FAM19A5 with a K _D of 10 nM or less as determined by an enzyme-linked immunosorbent assay (ELISA);
(e) binds to membrane-bound human FAM19A5 with a K _D of 10 nM or less as measured by ELISA;
(f) reduction, reversal, delay and/or prevention of reactive gliosis onset;
(g) suppression of hyperproliferation of reactive astrocytes;
(h) reduced expression of chondroitin sulfide proteoglycans, including neurocans and glial antigen 2 (NG2);
(i) increased expression of nuclear c-fos and pERK in neurons;
(j) promotion of neuronal cell survival;
(k) increased expression of intraneuronal GAP43; and (l) enhanced axonal regrowth.

In some embodiments, the anti-FAM19A5 antibody (eg, scFv) has a high affinity, eg, a K _D of 10 ⁻⁷ M or less, 10 ⁻⁸ M or less, 10 ⁻⁹ M (1 nM) or less, 10 ^{− 10} M (0.1 nM) or less, 10 ⁻¹¹ M or less, or 10 ⁻¹² M or less, such as 10 ⁻¹² M to 10 ⁻⁷ M, 10 ⁻¹¹ M to 10 ⁻⁷ M, 10 ⁻¹⁰ M to 10 ⁻⁷ M or 10 ⁻⁹ M to 10 ⁻⁷ M, such as 10 ⁻¹² M, 5×10 ⁻¹² M, 10 ⁻¹¹ M, 5×10 ⁻¹¹ M, 10 ⁻¹⁰ M, 5×10 ⁻¹⁰ M, 10 specifically binds to soluble human FAM19A5 or membrane-bound human that is ⁻⁹ M, 5×10 ⁻⁹ M, 10 ⁻⁸ M, 5×10 ⁻⁸ M, 10 ⁻⁷ M or 5×10 ⁻⁷ M . Standard analytical methods for assessing the binding ability of antibodies to human FAM19A5 of various species are known in the art, including, for example, ELISA, Western blot and RIA. The binding kinetics (eg, binding affinity) of the antibody may be assessed by standard analytical methods well known in the art, such as ELISA, BIACORE ^™ analysis or KINEXA®.

In some embodiments, the anti-FAM19A5 antibody (eg, scFv) has a K _D of 10 ⁻⁷ M or less, 10 ⁻⁸ M (10 nM) or less, 10 ⁻⁹ M (1 nM), eg, as determined by ELISA. 10 ⁻¹⁰ M or less, 10 ⁻¹² M to 10 ⁻⁷ M, 10 ⁻¹¹ M to 10 ⁻⁷ M, 10 ⁻¹⁰ M to 10 ⁻⁷ M, 10 ⁻⁹ M to 10 ⁻⁷ M, or 10 It binds to soluble human FAM19A5 from ^-8 M to 10 ^-7 M. In some embodiments, the anti-FAM19A5 antibody (eg, scFv) has a K _D of 10 nM or less, such as 0.1-10 nM, 0.1-5 nM, 0.1-1 nM, 0.5-10 nM, 0 Binds soluble FAM19A5 at .5-5 nM, 0.5-1 nM, 1-10 nM, 1-5 nM or 5-10 nM. In some embodiments, the anti-FAM19A5 antibody (e.g., scFv) has a K _D of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, as determined by ELISA. or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM or 90 nM specifically binds soluble human FAM19A5.

In some embodiments, the anti-FAM19A5 antibody (eg, scFv) has a K _D of 10 ⁻⁷ M or less, 10 ⁻⁸ M (10 nM) or less, 10 ⁻⁹ M (1 nM), eg, as determined by ELISA. 10 ⁻¹⁰ M or less, 10 ⁻¹² M to 10 ⁻⁷ M, 10 ⁻¹¹ M to 10 ⁻⁷ M, 10 ⁻¹⁰ M to 10 ⁻⁷ M, 10 ⁻⁹ M to 10 ⁻⁷ M or 10 ⁻ Binds membrane-bound human FAM19A5, which is ⁸ M to 10 ⁻⁷ M. In certain embodiments, the anti-FAM19A5 antibody (eg, scFv) has a K _D of 10 nM or less, eg, 0.1-10 nM, 0.1-5 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 0.1-1 nM, 5-10 nM, 0.5-5 nM, 0.5-1 nM, 1-10 nM, 1-5 nM or 5-10 nM specifically binds to membrane bound human FAM19A5. In some embodiments, the anti-FAM19A5 antibody (e.g., scFv) has a K _D of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, as determined by ELISA. or about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or about 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM or 90 nM, binds membrane bound human FAM19A5.

In some embodiments, an anti-FAM19A5 antibody (e.g., scFv) suitable for the methods disclosed herein is an anti-FAM19A5 antibody disclosed herein to bind (or inhibit binding) to a human FAM19A5 epitope. cross-compete with an anti-FAM19A5 antibody that contains the CDRs or variable regions of

In some embodiments, the anti-FAM19A5 antibody (e.g., scFv) inhibits binding of a reference antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, and (i) The heavy chain CDRl, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and the light chain CDRl, CDR2 and CDR3 of said reference antibody are respectively SEQ ID NO: 23, SEQ ID NO: 24 and the sequences (ii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 14, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 15, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 15; 16 amino acid sequences, wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:27, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:28. (iii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 17, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 18, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 19 said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:29, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:30, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:31; said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:22, said light chain CDR1 comprises , wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:32, said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34; (v) said heavy chain CDR1 comprises 89, wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:90, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:91, and said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:92 wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:93, said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:94; (vi) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:95 wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:96, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:97, and said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:98 , said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 99 and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 100; Said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 102, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 103, said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 104, said light chain CDR2 comprises , wherein said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 106; (viii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 107, and said heavy chain CDR2 comprises: 108, wherein said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 109, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 110, and said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 111 wherein said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:112; (ix) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:113 and said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:114 wherein said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 115; said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 116; said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 117; (x) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 119; said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 120; CDR3 comprises the amino acid sequence of SEQ ID NO: 121, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 122, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 123, said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: (xi) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 125, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 126, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 127 wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 128, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 129, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 130 (xii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 131, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 132, and said heavy chain CDR3 comprises , wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 134, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 135, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 136 (xiii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 137, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 138, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 139 wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 140, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 141, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 142; (xiv) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 143; said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 144; said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 145; chain CDR1 comprises the amino acid sequence of SEQ ID NO: 146, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 147, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 148; The chain CDR1 comprises the amino acid sequence of SEQ ID NO:149, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:150, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:151, and said light chain CDR1 comprises the sequence 152, wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:153, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:154. In some embodiments, the heavy chain CDR1, CDR2 and CDR3 of said reference antibody each comprise the CDR1, CDR2 and CDR3 sequences shown in Table 2, and the light chain CDR1, CDR2 and CDR3 of said reference antibody respectively are shown in Table 3. contains the CDR1, CDR2 and CDR3 sequences shown in .

In some embodiments, the reference antibody comprises (a) heavy and light chain variable region sequences comprising SEQ ID NOs:35 and 39, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs:36 and 40, respectively; (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 37 and 41, respectively; (d) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 42, respectively; (e) SEQ ID NOs: 155 and 42, respectively. (f) heavy and light chain variable region sequences comprising SEQ ID NOs: 156 and 167, respectively; (g) heavy and light chain variable regions comprising SEQ ID NOs: 157 and 168, respectively. (h) heavy and light chain variable region sequences comprising SEQ ID NOs: 158 and 169, respectively; (i) heavy and light chain variable region sequences comprising SEQ ID NOs: 159 and 170, respectively; (j) SEQ ID NOs: 160 and 160, respectively. (k) heavy and light chain variable region sequences comprising SEQ ID NOS: 161 and 172, respectively; (l) heavy and light chain variable region sequences comprising SEQ ID NOS: 162 and 173, respectively. sequences; (m) heavy and light chain variable region sequences comprising SEQ ID NOS: 163 and 174, respectively; (n) heavy and light chain variable region sequences comprising SEQ ID NOS: 164 and 175, respectively; or (o) SEQ ID NO: 165, respectively. and 176 heavy and light chain variable region sequences. In some embodiments, the reference antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises an amino acid sequence shown in Table 4, and said VL is shown in Table 5. including the amino acid sequence shown.

In some embodiments, the anti-FAM19A5 antibody (eg, scFv) reduces binding of such reference antibody to human FAM19A5 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%. , 80%, 90% or 100% inhibition. Competing antibodies bind to the same epitope, overlapping epitopes or adjacent epitopes (as evidenced by steric hindrance, for example). Whether two antibodies compete with each other for binding to a target can be determined using competition experiments known in the art, such as RIA and EIA.

In some embodiments, the anti-FAM19A5 antibody (e.g., scFv) binds to the same FAM19A5 epitope as a reference antibody disclosed herein, including heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3. (i) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 11, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 12, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 13; said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:23, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:24, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:25; (ii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:14, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:15, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:16, and said light chain CDR1 comprises: (iii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:26, said light chain CDR2 comprising the amino acid sequence of SEQ ID NO:27, said light chain CDR3 comprising the amino acid sequence of SEQ ID NO:28; 17, wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 18, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 19, and said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 29 wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:30, said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:31; (iv) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:20 wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:21, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:22, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:32, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:33 and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:34; (v) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:89; comprises the amino acid sequence of SEQ ID NO:90, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:91, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:92, and said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:93 and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:94; (vi) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO:95 and said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO:96 amino wherein said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO:97, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO:98, and said light chain CDR2 comprises the amino acid sequence of SEQ ID NO:99; (vii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 101; said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 102; heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 103, said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 104, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 105, and said light chain CDR3 comprises: (viii) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 107, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 108, and said heavy chain CDR3 comprises the sequence 109, wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 110, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 112 (ix) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 113, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 114, and said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 115 wherein said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 116, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 117, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 118; a) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 119, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 120, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 121, said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 122, said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 123, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 124; (xi) said heavy chain CDRl comprises , the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 126, the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 127, and the light chain CDRl comprises the amino acid sequence of SEQ ID NO: 128 (xii) said heavy chain C DR1 comprises the amino acid sequence of SEQ ID NO: 131, said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 132, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 133, and said light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 134, wherein said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 135, and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 136; (xiii) said heavy chain CDRl comprises SEQ ID NO: 137 wherein said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 138, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 139, and said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 140 , said light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 141 and said light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 142; (xiv) said heavy chain CDRl comprises the amino acid sequence of SEQ ID NO: 143; Said heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 144, said heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 145, said light chain CDRl comprises the amino acid sequence of SEQ ID NO: 146, said light chain CDR2 comprises or (xv) said heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 149 and said heavy chain CDR2 comprises , wherein the heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 151, the light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 152, and the light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 153 comprising an amino acid sequence, wherein said light chain CDR3 comprises the amino acid sequence of SEQ ID NO:154. In some embodiments, the heavy chain CDR1, CDR2 and CDR3 of said reference antibody each comprise the CDR1, CDR2 and CDR3 sequences shown in Table 2, and the light chain CDR1, CDR2 and CDR3 of said reference antibody respectively comprise the sequences shown in Table 3. contains the CDR1, CDR2 and CDR3 sequences shown in .

In some embodiments, the reference antibody comprises (a) heavy and light chain variable region sequences comprising SEQ ID NOs:35 and 39, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs:36 and 40, respectively; (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 37 and 41, respectively; (d) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 42, respectively; (e) SEQ ID NOs: 155 and 42, respectively. (f) heavy and light chain variable region sequences comprising SEQ ID NOs: 156 and 167, respectively; (g) heavy and light chain variable regions comprising SEQ ID NOs: 157 and 168, respectively. (h) heavy and light chain variable region sequences comprising SEQ ID NOs: 158 and 169, respectively; (i) heavy and light chain variable region sequences comprising SEQ ID NOs: 159 and 170, respectively; (j) SEQ ID NOs: 160 and 160, respectively. (k) heavy and light chain variable region sequences comprising SEQ ID NOS: 161 and 172, respectively; (l) heavy and light chain variable region sequences comprising SEQ ID NOS: 162 and 173, respectively. sequences; (m) heavy and light chain variable region sequences comprising SEQ ID NOS: 163 and 174, respectively; (n) heavy and light chain variable region sequences comprising SEQ ID NOS: 164 and 175, respectively; or (o) SEQ ID NO: 165, respectively. and 176 heavy and light chain variable region sequences. In some embodiments, the reference antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises an amino acid sequence shown in Table 4, and said VL is shown in Table 5. including the amino acid sequence shown.

Techniques for determining whether two antibodies bind to the same epitope include, for example, x-ray analysis of crystals of antigen:antibody complexes and hydrogen/deuterium exchange mass spectroscopy, which provide atomic resolution of the epitope. Epitope mapping methods such as analysis (HDX-MS), methods of monitoring antibody binding to antigen fragments or mutated variants of antigens in which loss of binding due to variations in amino acid residues within the antigen sequence is often taken as an indication of the epitope component. , including a computational combinatorial method for epitope mapping.

An anti-FAM19A5 antibody (eg, scFv) useful in the methods disclosed herein can bind to at least one epitope of mature human FAM19A5, eg, as determined by binding of the antibody to a fragment of human FAM19A5. In some embodiments, the anti-FAM19A5 antibody is a fragment located within the amino acid sequence of TLDRDSSQPRRTIARQTARC (amino acid residues 42-61 of SEQ ID NO:6 or SEQ ID NO:2), such as at least 1, 2 of SEQ ID NO:6, It binds to an epitope with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some embodiments, the anti-FAM19A5 antibody comprises amino acid residues 46-51 (ie, DSSQPR), such as amino acid residues 46, 50 and 52 (ie, D---PR), such as SEQ ID NO:2. Binds to SEQ ID NO:6 at one or more amino acids corresponding to amino acid residues 46, 47, 48 and 50 (ie, DSS-P). In some embodiments, the anti-FAM19A5 antibody is a fragment (amino acids 90-109 of SEQ ID NO:9 or SEQ ID NO:2) located within the amino acid sequence of CDMLPCLEGEGCDLLLINRSG, such as at least 1, 2, 3, 4 of SEQ ID NO:9. , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In certain embodiments, the anti-FAM19A5 antibody comprises one or more amino acid residues 99-107 (ie, EGCDLLLINR), such as amino acid residues 102, 103, 105 and 107 (ie, DL-IR), such as amino acid residues 99, 100, 102, 103, 105 and 107 (ie EG-DL-IR), such as amino acid residues 99, 100 and 107 of SEQ ID NO: 4 (ie EG------R) to SEQ ID NO:9.

In some embodiments, the at least one epitope is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of SEQ ID NO:6 have the same amino acid sequence. In some embodiments, the at least one epitope is at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% have the same amino acid sequence.

In some embodiments, the anti-FAM19A5 antibody (e.g., scFv) is a human FAM19A5 epitope that is SEQ ID NO: 5, 6, 7, 8, 9 or 10 or SEQ ID NO: 5, 6, 7, 8, 9 or 10 e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of SEQ ID NO: 5, 6, 7, 8, 9 or 10 , 16, 17, 18, 19 or 20 amino acids.

In some embodiments, the anti-FAM19A5 antibodies of the present disclosure bind to SEQ ID NO:6 or a fragment thereof in its native conformation (ie, native). In some embodiments, the anti-FAM19A5 antibody binds to SEQ ID NO:9 or a fragment thereof in its native conformation (ie, native). In some embodiments, the anti-FAM19A5 antibody binds to both glycosylated and non-glycosylated human FAM19A5.

In some embodiments, the anti-FAM19A5 antibody (eg, scFv) further binds to one or more additional FAM19A5 epitopes. Thus, a particular anti-FAM19A5 antibody binds to (i) the epitope of SEQ ID NO:6 and additional epitopes, or (ii) the epitope of SEQ ID NO:9 and additional epitopes. Other anti-FAM19A5 antibodies can bind epitopes of SEQ ID NO:5, SEQ ID NO:9 and additional epitopes. In some embodiments, the anti-FAM19A5 antibody binds to the epitope of SEQ ID NO:6, the epitope of SEQ ID NO:10 and additional epitopes.

In some embodiments, the one or more additional FAM19A5 epitopes are QLAAGTCEIVTLDR (SEQ ID NO:5, epitope F1), TLDRDSSQPRRTIARQTARC (SEQ ID NO:6, epitope F2), TARCACRKGQIAGTTRARPA (SEQ ID NO:7, epitope F3), ARPACVDARIIKTKQWCDML (sequence No. 8, epitope F4), CDMLPCLEGEGCDLLINRSG (SEQ ID No. 9, epitope F5) or NRSGWTCTQPGGRIKTTTVS (SEQ ID No. 10, epitope F6) or SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No. 10 or any combination thereof. Fragments located within the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 are SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8, sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of No. 9 or SEQ ID No. 10 contains fragments with In some embodiments, the one or more additional FAM19A5 epitopes are within the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9 or 10, or SEQ ID NO: 5, 6, 7, 8, 9 or 10. positioned fragments such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of SEQ ID NO: 5, 6, 7, 8, 9 or 10; Selected from fragments having 16, 17, 18, 19 or 20 amino acids, or any combination thereof. In some embodiments, an anti-FAM19A5 antibody of the disclosure binds to an epitope of any of the one or more additional epitopes in its native conformation (ie, native). In some embodiments, the anti-FAM19A5 antibody binds to both one or more of the glycosylated and non-glycosylated FAM19A5 epitopes.

In some embodiments, an anti-FAM19A5 antibody (e.g., scFv) of the present disclosure binds to at least one FAM19A5 epitope identified as EP2, EP4 and/or EP8, wherein EP2 comprises amino acids DSSQP (SEQ ID NO: 66 ), EP4 comprises, consists essentially of, or consists of, the amino acid ARCACRK (SEQ ID NO: 68), EP8 comprises, consists essentially of, or consists of the amino acids TCTQPGGR (SEQ ID NO:72). In some embodiments, the at least one epitope is EP2, EP4 or EP8 and at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about They have 100% identical amino acid sequences. In some embodiments, only the anti-FAM19A5 antibody binds EP2. In some embodiments, the anti-FAM19A5 antibody binds to EP4 and EP8.

In some embodiments, the anti-FAM19A5 antibody binds to at least one FAM19A5 epitope identified as EP6, EP7 or EP8, EP6 comprising amino acids KTKQWCDML (SEQ ID NO: 70) and EP7 comprising amino acids GCDLLLINR ( 71) and EP8 contains the amino acids TCTQPGGR (SEQ ID NO:72). In some embodiments, the at least one epitope is EP6, EP7 or EP8 and at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about They have 100% identical amino acid sequences. In some embodiments, the anti-FAM19A5 antibody only binds EP6, EP7 or EP8. In some embodiments, the anti-FAM19A5 antibody binds to EP6, EP7 and EP8. In some embodiments, the anti-FAMT9A5 antibody binds to EP7 and EP8. In some embodiments, the anti-FAM19A5 antibody binds EP7.

In some embodiments, the anti-FAM19A5 antibody is from SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and any combination thereof binds to one or more FAM19A5 epitopes selected from the group consisting of

In some embodiments, the present disclosure provides 20%, 25%, 30% relative to other proteins in the FAM19A family, as measured by, for example, immunoassays (e.g., ELISA), surface plasma resonance or dynamic exclusion assays. %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity of FAM19A5 (e.g. Antibodies that bind to human FAM19A5) are provided. In certain embodiments, the description provides antibodies that bind FAM19A5 (eg, human FAM19A5) without cross-reactivity with yet other proteins in the FAM19A family, eg, as determined by immunoassay.

In some embodiments, the anti-FAM19A5 antibody is not a natural antibody or a naturally occurring antibody. For example, the anti-FAM19A5 antibody has post-translational modifications that differ from naturally occurring antibodies, eg, by having more, fewer, or different types of post-translational modifications from each other.

<Exemplary Anti-FAM19A5 Antibodies>
Particular antibodies that can be expressed by the AAV vectors disclosed herein are antibodies having the CDR and/or variable region sequences of anti-FAM19A5 antibodies 1-65, 3-2 and 2-13, such as scFv, and variable regions thereof Or an antibody with at least 80% identity (eg, at least 85%, at least 90%, at least 95%, or 99% or more identity) to the CDR sequences. See US Patent No. 9,579,398; International Application No. PCT/IB2017/001490. Tables 2 and 3 provide exemplary heavy and light chain CDR sequences, respectively. CDRs for the following antibodies were identified using the Kabat numbering system (see above): 1-65, 3-2, 2-13, 1-28, P2-C12, 13B4, 13F7, 15A9, Pl-A03, P1-A08, P1-F02, P2-A01, P2-A03, P2-F07, P2-F11, SS01-13, SS01-13-s5 and S5-2. GKNG. CDRs for the following antibodies were identified using the IMGT numbering system (see above): 1-7A-IT, Low-PI, 1-30, 1-17, 1-32, 4-11, 6-10, 2-13D, 2-13D-37, 2-13D-37-1.5W-41 and 2-13D-37-3W-16. The VH and VL amino acid sequences of individual anti-FAM19A5 antibodies of this disclosure are provided in Tables 4 and 5, respectively.

In some embodiments, the anti-FAM19A5 antibody comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NOs: 35-38, 155-165 or 232-240 ( See Table 4). In another embodiment, the anti-FAM19A5 antibody comprises heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 35-38, 155-165 or 232-240 (see Table 4).

In some embodiments, the anti-FAM19A5 antibody comprises heavy and light chain variable regions, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NOs: 39-42, 166-176 or 241-250 (Table 5). In another embodiment, the anti-FAM19A5 antibody comprises light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 39-42, 166-176 or 241-250 (see Table 5).

In some embodiments, the anti-FAMT9A5 antibody comprises a heavy chain variable region CDR (see Table 4) selected from the group consisting of SEQ ID NOs: 35-38, 155-165 or 232-240 and SEQ ID NOs: 39-42 , 166-176 or 241-250 (see Table 5).

In some embodiments, the anti-FAM19A5 antibody comprises heavy and light chain variable regions, (i) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37, and said light chain variable region comprises the sequence (ii) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO:36 and said light chain variable region comprises the amino acid sequence of SEQ ID NO:40; (iii) said heavy chain variable a region comprising the amino acid sequence of SEQ ID NO:35, said light chain variable region comprising the amino acid sequence of SEQ ID NO:39; (iv) said heavy chain variable region comprising the amino acid sequence of SEQ ID NO:38, said the variable region comprises the amino acid sequence of SEQ ID NO: 42; (v) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 155 and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 166; (vii) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 156; and (vii) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 157; (viii) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 168; and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 169 (ix) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 159 and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 170; (x) said heavy chain variable region comprises SEQ ID NO: 160 amino acid sequence, said light chain variable region comprising the amino acid sequence of SEQ ID NO: 171; (xi) said heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 161, said light chain variable region comprising the sequence (xii) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 162; and said light chain variable region comprises the amino acid sequence of SEQ ID NO: 173; (xiii) said heavy chain variable (xiv) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 164, said light chain the variable region comprises the amino acid sequence of SEQ ID NO:175; and (xv) said heavy chain variable region comprises the amino acid sequence of SEQ ID NO:165 and said light chain variable region comprises the amino acid sequence of SEQ ID NO:176. In some embodiments, the isolated anti-FAM19A5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein said VH comprises a VH sequence shown in Table 4. , said VL comprises the VL sequences shown in Table 5.

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence set forth in SEQ ID NOs: 35-38, 155-165 or 232-240 (Table 1). 4) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% the same amino acid sequence as including.

In some embodiments, the anti-FAM19A5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region has the amino acid sequence set forth in SEQ ID NOs: 39-42, 166-176 or 241-250 ( See Table 5) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical amino acids Contains arrays.

In some embodiments, the anti-FAM19A5 antibody comprises heavy and light chain variable regions, wherein the heavy chain variable regions have the amino acid sequences set forth in SEQ ID NOs: 35-38, 155-165 or 232-240 (see Table 4). ) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% the same amino acid sequence as , said light chain variable region is at least about 80%, at least about 85%, at least about 90%, at least about 95% the amino acid sequence set forth in SEQ ID NOs: 39-42, 166-176 or 241-250 (see Table 5) , at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical amino acid sequences.

In some embodiments, the anti-FAM19A5 antibody is:
(a) heavy and light chain variable region sequences comprising SEQ ID NOs: 35 and 39, respectively;
(b) heavy and light chain variable region sequences comprising SEQ ID NOs: 36 and 40, respectively;
(c) heavy and light chain variable region sequences comprising SEQ ID NOs: 37 and 41, respectively;
(d) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 42, respectively;
(e) heavy and light chain variable region sequences comprising SEQ ID NOS: 155 and 166, respectively;
(f) heavy and light chain variable region sequences comprising SEQ ID NOs: 156 and 167, respectively;
(g) heavy and light chain variable region sequences comprising SEQ ID NOS: 157 and 168, respectively;
(h) heavy and light chain variable region sequences comprising SEQ ID NOS: 158 and 169, respectively;
(i) heavy and light chain variable region sequences comprising SEQ ID NOs: 159 and 170, respectively;
(j) heavy and light chain variable region sequences comprising SEQ ID NOs: 160 and 171, respectively;
(k) heavy and light chain variable region sequences comprising SEQ ID NOs: 161 and 172, respectively;
(l) heavy and light chain variable region sequences comprising SEQ ID NOs: 162 and 173, respectively;
(m) heavy and light chain variable region sequences comprising SEQ ID NOS: 163 and 174, respectively;
(n) heavy and light chain variable region sequences comprising SEQ ID NOS: 164 and 175, respectively;
(o) heavy and light chain variable region sequences comprising SEQ ID NOS: 165 and 176, respectively;
(p) heavy and light chain variable region sequences comprising SEQ ID NOS: 232 and 241, respectively;
(q) heavy and light chain variable region sequences comprising SEQ ID NOs: 233 and 242, respectively;
(r) heavy and light chain variable region sequences comprising SEQ ID NOs: 234 and 242, respectively;
(s) heavy and light chain variable region sequences comprising SEQ ID NOs: 235 and 243, respectively;
(t) heavy and light chain variable region sequences comprising SEQ ID NOs: 236 and 244, respectively;
(u) heavy and light chain variable region sequences comprising SEQ ID NOS: 236 and 245, respectively;
(v) heavy and light chain variable region sequences comprising SEQ ID NOs: 236 and 246, respectively;
(w) heavy and light chain variable region sequences comprising SEQ ID NOs: 236 and 247, respectively;
(x) heavy and light chain variable region sequences comprising SEQ ID NOs: 236 and 248, respectively;
(y) heavy and light chain variable region sequences comprising SEQ ID NOs: 236 and 249, respectively;
(z) heavy and light chain variable region sequences comprising SEQ ID NOs: 237 and 250, respectively;
(aa) heavy and light chain variable region sequences comprising SEQ ID NOs: 238 and 250, respectively;
(bb) heavy and light chain variable region sequences comprising SEQ ID NOs:239 and 250, respectively; or (cc) heavy and light chain variable region sequences comprising SEQ ID NOs:240 and 250, respectively.

In certain embodiments, the anti-FAM19A5 antibody comprises (i) 1-65 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 1-65 light chain CDR1, CDR2 and CDR3 or combinations thereof; ii) 3-2 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 3-2 light chain CDR1, CDR2 and CDR3 or any combination thereof; (iii) 2-13 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 2-13 light chain CDR1, CDR2 and CDR3 or any combination thereof; (iv) 1-28 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 1- 28 light chain CDR1, CDR2 and CDR3 or any combination thereof; (v) P2-C12 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or P2-C12 light chain CDR1, CDR2 and CDR3 or any combination thereof; (vi) heavy chain CDR1, CDR2 and CDR3 of 13B4 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of 13B4 or any combination thereof; (vii) heavy chain CDR1, CDR2 of 13F7 and CDR3 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of 13F7 or any combination thereof; (viii) heavy chain CDR1, CDR2 and CDR3 of 15A9 or combinations thereof and/or light chain CDR1 of 15A9; (ix) the heavy chain CDR1, CDR2 and CDR3 of P1-A03 or combinations thereof and/or the light chain CDR1, CDR2 and CDR3 of P1-A03 or any combination thereof; x) heavy chain CDR1, CDR2 and CDR3 of P1-A08 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of P1-A08 or any combination thereof; (xi) heavy chain CDR1, CDR2 of P1-F02 and CDR3 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of P1-F02 or any combination thereof; (xii) heavy chain CDR1, CDR2 and CDR3 of P2-A01 or combinations thereof and/or P2- (xiii) the heavy chain CDR1, CDR2 and CDR3 of P2-A03 or combinations thereof and/or the light chain CDR1 of P2-A03; (xiv) the heavy chain CDR1, CDR2 and CDR3 of P2-F07 or combinations thereof and/or the light chain CDR1, CDR2 and CDR3 of P2-F07 or any combination thereof; xv) heavy chain CDR1, CDR2 and CDR3 of P2-F11 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of F2-F11 or any combination thereof; (xvi) heavy chain CDR1, CDR2 of SS01-13 and CDR3 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of SS01-13 or any combination thereof; (xvii) heavy chain CDR1, CDR2 and CDR3 of SS01-13-s5 or combinations thereof and/or the light chain CDR1, CDR2 and CDR3 of SS01-13-s5 or any combination thereof; (xviii) S5-2. GKNG heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or S5-2. light chain CDR1, CDR2 and CDR3 of GKNG or any combination thereof; (xix) heavy chain CDR1, CDR2 and CDR3 of 1-7A-IT or combinations thereof and/or light chain CDR1, CDR2 of 1-7A-IT and CDR3 or any combination thereof; (xx) Low-PI heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or Low-PI light chain CDR1, CDR2 and CDR3 or any combination thereof; (xxi ) 1-30 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 1-30 light chain CDR1 , CDR2 and CDR3 or any combination thereof; (xxii) 1-17 heavy chain CDR1 , CDR2 and (xxiii) heavy chain CDR1, CDR2 and CDR3 of 1-32 or combinations thereof and/or 1-32 (xxiv) the heavy chain CDR1, CDR2 and CDR3 of 4-11 or combinations thereof and/or the light chain CDR1, CDR2 and CDR3 of 4-11 or any combination thereof; (xxv) 6-10 heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or 6-10 light chain CDR1, CDR2 and CDR3 or any combination thereof; (xxvi) 2-13D heavy chain CDR1, CDR2 and CDR3 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of 2-13D or any combination thereof; (xxvii) heavy chain CDR1, CDR2 and CDR3 of 2-13D-37 or these and/or light chain CDR1, CDR2 and CDR3 of 2-13D-37 or any combination thereof; (xxviii) heavy chain CDR1, CDR2 and CDR3 of 2-13D-37-1.5W-41 or any combination thereof; combination and/or light chain CDR1, CDR2 and CDR3-41 of 2-13D-37-1.5W-41 or any combination thereof; or (xxiv) heavy chain CDR1 of 2-13D-37-3W-16, CDR2 and CDR3 or combinations thereof and/or light chain CDR1, CDR2 and CDR3 of 2-13D-3W-16 or combinations thereof. The amino acid sequences of VH CDR1, CDR2 and CDR3 for individual anti-FAM19A5 antibodies disclosed herein are provided in Table 2. The amino acid sequences of VL CDR1, CDR2 and CDR3 for individual anti-FAM19A5 antibodies disclosed herein are provided in Table 3.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:17; and/or (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:18; and/or (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VH CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:29; and/or (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:30; and/or (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:31.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VL CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 18;
(c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:29;
(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:30; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:31.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:15; and/or (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:16.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VH CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:26;
(b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and/or (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:28.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VL CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 14;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 15;
(c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 16;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:26;
(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:28.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 11;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:12; and/or (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:13.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VH CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:23;
(b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:24; and/or (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VL CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 11;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12;
(c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 13;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:23;
(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:24; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:20;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:21; and/or (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VH CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:32;
(b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:33; and/or (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, said anti-FAM19A5 antibody comprises any one, two or three of said VL CDRs.

In some embodiments, the anti-FAM19A5 antibody is:
(a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:20;
(b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:21;
(c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:22;
(d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:32;
(e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:33; and/or (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:34.

In some embodiments, an anti-FAM19A5 antibody (eg, scFv) disclosed herein comprises 1, 2, 3, 4, 5, or 6 of said CDRs. In some embodiments, the anti-FAM19A5 antibodies (e.g., scFv) disclosed herein have 1, 2, 3, 4, 5 or 6 of the CDRs provided in Table 2 or Table 3. Including pcs.

In some embodiments, the VH and VL domains described herein can be joined by a flexible linker to produce single chain Fv antibodies. Accordingly, in some embodiments, the anti-FAM19A5 antibody or antigen-binding portion thereof comprises a VH domain selected from Table 4 and a VL domain selected from Table 5, wherein the VH and VL domains are flexible joined by a linker. In some embodiments, the flexible linker comprises the sequence SGGGGSSGGGGS (SEQ ID NO:207). In another embodiment, the flexible linker comprises the sequence GQSSRSSGGGGSSGGGGS (SEQ ID NO:300). The amino acid sequence of an exemplary anti-FAM19A5 scFv is provided in Table 6 below (VH domains are underlined, VL domains are shown in bold, linkers are shown in italics).

A VH domain or one or more CDRs thereof as described herein may also be joined to a constant domain to form a heavy chain, eg, a full length heavy chain. Similarly, a VL domain, or one or more CDRs thereof, as described herein may be joined to a constant domain to form a light chain, eg, a full-length light chain. The full-length heavy chain and full-length light chain are combined to produce a full-length antibody.

Thus, in certain embodiments, antibodies are provided that comprise antibody light and heavy chains, eg, distinct light and heavy chains useful in the methods disclosed herein. With respect to said light chains, in certain embodiments, the light chains of the antibodies described herein are kappa light chains. In other specific embodiments, the light chains of the antibodies described herein are lambda light chains. In yet other specific embodiments, the light chains of the antibodies described herein are human kappa light chains or human lambda light chains. In certain embodiments, antibodies useful in the methods disclosed herein that specifically bind to a FAM19A5 polypeptide (e.g., human FAM19A5) bind any VL or VL CDR amino acid sequence described herein. wherein the constant region of said light chain comprises the amino acid sequence of a human kappa light chain constant region. In certain embodiments, antibodies useful in the methods disclosed herein that specifically bind to a FAM19A5 polypeptide (eg, human FAM19A5) are VL or VL CDR amino acid sequences described herein. The constant region of said light chain comprises the amino acid sequence of a human lambda light chain constant region. Non-limiting examples of human constant region sequences are described in the art. See, for example, US Pat. No. 5,693,780 and Kabat EA et al, (1991) above.

In some embodiments, the heavy chains of the antibodies described herein are alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ). It can be a heavy chain. In certain other embodiments, the heavy chains of the described antibodies can comprise human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chains. In some embodiments, an antibody useful in the methods disclosed herein that specifically binds to FAM19A5 (eg, human FAM19A5) is a heavy chain comprising a VH or VH CDR amino acid sequence described herein. wherein the heavy chain constant region comprises the amino acid sequence of a human gamma (γ) heavy chain constant region. In other embodiments, an antibody described herein that specifically binds FAM19A5 (e.g., human FAM19A5) comprises a heavy chain comprising a VH or VH CDR amino acid sequence described herein, said heavy chain The constant region of the chain comprises amino acids of a human heavy chain as described herein or known in the art. Non-limiting examples of human constant region sequences are described in the art. For example, US Pat. No. 5,693,780 and Kabat EA et al., above. , (1991).

In some embodiments, antibodies useful in the methods disclosed herein comprise VL and VH domains comprising the VH or VH CDRs and VL and VL CDRs described herein, wherein the constant region is , IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules or the constant region amino acid sequences of human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules. In other specific embodiments, antibodies described herein that specifically bind to FAM19A5 (e.g., human FAM19A5) comprise VL and VH domains comprising any of the amino acid sequences described herein. , said constant region is the amino acid sequence of the constant region of IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecules and immunoglobulin molecules of any subtype (e.g. IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) including. In some embodiments, the constant region is a native human IgG, including subtypes (eg, IgG1, IgG2, IgG3 or IgG4) and allogeneic types (eg, Glm, G2m, G3m and nG4m) and variants thereof. contains the amino acid sequence of the constant region of For example, Vidarsson G.; et al. Front Immunol. 5:520 (published online October 20, 2014) and Jefferis R. and Lefranc MP, mAbs 1:4, 1-7 (2009). In some embodiments, the constant region comprises the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 or variant thereof constant region.

In certain embodiments, anti-FAM19A5 antibodies useful in the methods disclosed herein exhibit Fc effector function, e.g., complement dependent cytotoxicity (CDC) and/or antibody-dependent cellular phagocytosis (ADCP). ). Effector functions are mediated by the Fc region, and the residues in the CH2 domain of the Fc region closest to the hinge region are responsible for C1q (complement) and IgG-Fc receptors ( FcγR) and thus are responsible for antibody effector functions. IgG2 and IgG4 antibodies also have lower levels of Fc effector function than IgG1 and IgG3 antibodies. The effector functions of antibodies are characterized by: (1) antibody fragments lacking the Fc region (such as Fab, F(ab')2, single chain Fv (scFv) or sdAbs consisting of monomeric VH or VL domains); (2) antibody in cells cultured in the presence of a glycosylation inhibitor by enzymatically removing the sugar, for example by deleting or altering the residue to which the sugar is attached; or by expressing the antibody in cells that are incapable of glycosylating proteins (e.g., bacterial host cells, see, e.g., U.S. Patent Publication No. 20120100140); (3) effector function; using Fc regions of IgG subtypes with reduced IgG (e.g., regions of IgG2 and IgG4 antibodies or chimeric Fc regions comprising the CH2 domains of IgG2 and IgG4 antibodies, e.g., US Patent Publication No. 20120100140 and Lau C. et al.J. 191:4769-4777 (2013)); and (4) reduction or avoidance by different approaches known in the art, including generating Fc regions with mutations that reduce or eliminate Fc function. can be See, eg, US Patent Publication No. 20120100140 and US and PCT applications cited therein and An et al, mAbs 1:6,572-579 (2009).

Thus, in some embodiments, anti-FAM19A5 antibodies useful in the methods disclosed herein are Fab, Fab', F(ab')2, Fv, single chain Fv (scFv) or monomeric VH or an sdAb consisting of a VL domain. Such antibody fragments are well known in the art and are described above.

In some embodiments, anti-FAM19A5 antibodies useful in the methods disclosed herein comprise an Fc region with reduced or absent Fc effector function. In some embodiments, the constant region comprises the amino acid sequence of a human IgG2 or IgG4 Fc region, and in some embodiments, the anti-FAM19A5 antibody has an IgG2/IgG4 isotype. In some embodiments, the anti-FAM19A5 antibody comprises a chimeric Fc region comprising the CH2 domain of an IgG antibody of the IgG4 isotype and the CH3 domain of an IgG antibody of the IgG1 isotype, or an IgG2 hinge region and an IgG4 CH2 region. Chimeric Fc regions, or Fc regions with mutations in which Fc effector function is reduced or absent. Fc regions with reduced or absent Fc effector function include those known in the art. For example, Lau C. et al,J. Immunol. 191:4769-4777 (2013); An et al, mAbs 1:6, 572-579 (2009); and US Patent Publication No. 20120100140 and US patents and publications and PCT publications cited therein. sea bream. In addition, Fc regions with reduced or absent Fc effector function can be readily produced by those skilled in the art.

<Nucleic acid molecule>
Another aspect described herein pertains to one or more nucleic acid molecules encoding any one of the antibodies or antigen-binding portions thereof disclosed herein. Such nucleic acid molecules can be expressed in the AAV vectors of this disclosure. Nucleic acids are removed from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to naturally isolated DNA) or proteins by alkaline/SDS treatment, CsCl "isolated" or "substantially pure" when purified by standard techniques, including banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. ”. F. Ausubel, et al. , ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein are, for example, DNA or RNA and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

Particular nucleic acid molecules described herein encode the VH and VL sequences of the anti-FAM19A5 antibodies of this disclosure. Exemplary DNA sequences encoding VH sequences for such antibodies are shown in SEQ ID NOS: 43-46, 177 and 265-278 (Table 7). Exemplary DNA sequences encoding VL sequences for such antibodies are shown in SEQ ID NOS: 47-50, 178 and 279-292 (Table 8). Exemplary DNA sequences encoding the 1-65, 3-2 and 2-13 scFv antibodies are provided in Table 9.

To generate the anti-FAM19A5 scFv antibodies disclosed herein, the VH- and VL-encoding DNA fragments are operatively attached to a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3. and the VH and VL sequences can be expressed as contiguous single-chain proteins in which the VL and VH regions are joined by a flexible linker (eg Bird et al, (1988) Science 242:423-426; Huston USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554). Such nucleic acid sequences can then be inserted into the AAV vectors disclosed herein, which can later be used to produce scFv antibodies, eg, as demonstrated in the Examples.

Alternatively, nucleotide sequences encoding the heavy and light chains of said anti-FAM19A5 antibody (with or without signal peptide), e.g. , SEQ ID NOs:46 and 50, SEQ ID NOs:177 and 178 can be inserted into one or more AAV vectors of the present disclosure. Such AAV vectors can then be used to produce full-length antibodies. Included herein are host cells transduced with the AAV vectors of the present disclosure.

Methods of producing the AAV vectors disclosed herein are well known in the art, including, for example, methods using packaging cells, auxiliary viruses or plasmids and/or baculovirus systems (see, for example, Samulski et al. al., J. Virology 63, 3822 (1989);Xiao et al., J. Virology 72, 2224 (1998); 072364).

In addition to methods for producing pseudotyped AAV vectors (e.g., WO00/28004), various modifications or formulations of AAV vectors are used to reduce the immunogenicity of AAV vectors when administered in vivo. are known (see, for example, WO01/23001; WO00/73316; WO04/112727; WO05/005610; and WO99/06562). In some embodiments, AAV vectors can be produced or derived from different serotypes of AAV that can be mixed together or mixed with other types of virus to produce chimeric (e.g., pseudotyped) AAV viruses. can.

<III. Pharmaceutical composition>
The present specification provides the desired degree of purity in physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Compositions comprising AAV antibodies are provided. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffering agents such as phosphates, citrates and other organic acids; ascorbic acid and methionine. antioxidants; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sucrose, mannitol, trehalose or sorbitol. salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG). .

In some embodiments, pharmaceutical compositions comprise an AAV vector described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition comprises, in a pharmaceutically acceptable carrier, an effective amount of an antibody or antigen-binding portion thereof described herein and optionally one or more additional prophylactic or therapeutic agents. containing agents. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be useful for reducing FAM19A5 activity and treating conditions such as central nervous system injury, disease or disorder, eg, neuropathic pain.

Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, tonicity agents, buffers, antioxidants, topical anesthetics, suspending and dispersing agents, emulsifying agents , sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Injection Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactate Injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed, corn, sesame and peanut oils. Multidose antimicrobial agents in bacteriostatic or fungistatic concentrations include phenol or cresol, mercurial-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. It can be added to parenteral formulations packaged in containers. Tonicity agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The pharmaceutical compositions of this disclosure can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, parenteral, intrathecal, intracerebroventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular or intravenous injection, is also contemplated herein. Injectables can be in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility-enhancing agents and, for example, sodium acetate, sorbitan monolaurate, Formulations such as triethanolamine oleate and cyclodextrin can be included.

Formulations for parenteral administration of antibodies include sterile dry soluble products such as instant sterile liquids for injection, lyophilized powders ready for extemporaneous reconstitution with solvents immediately prior to use, including tablets for subcutaneous injection, instant sterile suspensions for injection, Includes sterile dry insoluble products and sterile emulsions ready for extemporaneous formulation with vehicle immediately prior to use. Said solution may be aqueous or non-aqueous.

For intravenous administration, suitable carriers include saline or phosphate-buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof. include.

Antibody-containing topical mixtures are prepared as described for local and systemic administration. The resulting mixtures can be solutions, suspensions, emulsions, etc., including creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations. It can be formulated as an irrigation, spray, suppository, bandage, skin patch or any other dosage form suitable for topical administration.

The AAV antibodies described herein can be formulated, for example, as a topical aerosol for inhalation (see, for example, US Pat. No. 4,324,932, which describes steroid delivery aerosols useful for treating inflammatory diseases, particularly asthma). 044,126, 4,414,209 and 4,364,923). These dosage forms for respiratory tract administration are in the form of aerosols for nebulizers or solutions, or fine powders for inhalants, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the dosage form have diameters of less than 50 microns in some embodiments, and less than 10 microns in some embodiments.

The AAV antibodies described herein can be used topically or topically, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions, and for application or intracisternal to the eye. or can be formulated for intraspinal application. Topical administration is also contemplated for transdermal delivery and for ocular or mucosal administration or for inhalation therapy. Nasal solutions of the antibodies can be administered alone or in combination with other pharmaceutically acceptable excipients.

In some embodiments, pharmaceutical compositions comprising AAV antibodies described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions and other mixtures. The lyophilized powder can also be reconstituted and formulated as a solid or gel. The lyophilized powder comprises an antibody or antigen-binding portion thereof, or a pharmaceutically acceptable derivative thereof, as described herein dissolved in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solution made from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable formulation. The solvent can also contain a buffer such as citrate, sodium phosphate or potassium phosphate, or other such buffer at about neutral pH in some embodiments. The solution is then sterile filtered and then lyophilized under standard conditions known to those of skill in the art to provide the desired dosage form. In some embodiments, the resulting solution can be apportioned into lyophilization vials. Each vial can contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature.

Such lyophilized powders are reconstituted with water for injection to provide a dosage form for use for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount will depend on the compound chosen. Such amounts can be determined empirically.

The VVA antibodies described herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other body regions to be treated. Many such targeting methods are well known to those of skill in the art. Any such targeting methods are contemplated for use with the present compositions herein. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652; 6,274,552; , 139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039 , 975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 No. 5,709,874. In certain embodiments, the AAV vectors described herein are targeted to treat diseases or disorders, such as central nervous system injury, degenerative brain disease, or neuropathic pain.

A composition to be used for in vivo administration can be sterile. This may be facilitated by filtration, eg, using sterile filtration membranes.

<IV. Kit>
Also provided herein are kits comprising one or more of the AAV antibodies described herein. In some embodiments, this specification is satisfied by any one or more of the components of the pharmaceutical compositions described herein, such as one or more AAV antibodies provided herein. A pharmaceutical pack or kit is provided comprising one or more containers and optionally instructions for use. In some embodiments, the kit contains a pharmaceutical composition described herein and any prophylactic or therapeutic agent as described herein.

<V. Therapeutic use and method>
In certain aspects, the specification provides a method for reducing injury or damage to the CNS in a subject, comprising administering to said subject an AAV vector of the present disclosure.

In another aspect, the present specification provides a method for inhibiting, delaying, suppressing, limiting, reducing, reversing or preventing the onset or initiation of gliosis and its associated CNS detrimental effects in a subject, A method is provided comprising administering to a subject an AAV vector disclosed herein. In some embodiments, the present disclosure inhibits, slows, suppresses, limits, reduces, reverses, or prevents excessive or abnormal proliferation of reactive astrocytes and associated CNS detrimental effects in a subject. A method is provided comprising administering to said subject an AAV vector of the present disclosure. In some embodiments, the disclosure provides for reducing, inhibiting or alleviating the expression of chondroitin sulfide proteoglycans (including levels of neurocan, NG2 or both) in a subject, or reducing the activity of neurocan, NG2 or both. A method is presented comprising administering to said subject an AAV vector described herein as a method for reducing or inactivating. In some embodiments, the present disclosure provides an AAV vector as described herein in a subject as a method for stimulating, promoting, increasing or activating neuronal cell growth, preferably after injury or injury in said subject. A method is presented comprising administering a In other embodiments, the disclosure provides for increasing levels of c-fos mRNA, c-fos protein or c-fos protein activity in a subject in need thereof, preferably nuclear ERK mRNA, ERK protein in neuronal cells. or, as a method for increasing the level of pERK activity, a method comprising administering to said subject an AAV vector of the present disclosure. In certain embodiments, the present description provides a method for increasing or increasing the level of GAP43 mRNA, GAP43 protein in a subject in need thereof, or increasing the activity of GAP43 protein, preferably in a neuronal cell. A method is provided that includes administering an AAV vector of the present disclosure. In certain embodiments, the present disclosure provides a subject with the AAV disclosed herein as a method for enhancing or promoting neuronal survival and/or promoting axonal regrowth in a subject in need thereof. A method is presented that includes administering the vector. In some embodiments, the subject is a human, preferably a neuronal cell from, for example, CNS injury, trauma, injury, cerebrospinal injury, brain tumor, infection, ischemia, stroke, autoimmune reaction and/or neurodegenerative disease. are injured or injured humans.

In some aspects, the present description also provides a disease or disorder involving central nervous system injury, cerebrospinal injury, degenerative encephalopathy, degenerative cerebrospinal or neuropathy or neuropathic pain in a subject in need thereof. A method for treating is provided comprising administering to said subject an AAV vector of the present disclosure. In some embodiments, the central nervous system injury is traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof. In some embodiments, the degenerative brain disorder is Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), or a combination thereof. Hereby, in certain embodiments, the present specification provides a method for treating traumatic brain injury, cerebrospinal injury, stroke, brain tumor, or a combination thereof in a subject in need thereof. Disclosed are methods comprising administering the AAV vectors disclosed in , or compositions thereof. In some embodiments, the present disclosure provides a method for treating Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS in a subject in need of the AAV disclosed herein. Disclosed are methods comprising administering the vector or composition thereof. In some embodiments, the subject is human.

In certain embodiments, the present description provides a method for treating, reducing, reversing, preventing, alleviating, ameliorating or inhibiting fibrosis in a subject in need thereof, the AAV vector disclosed herein or A method is presented that includes administering the composition. In some embodiments, the fibrosis is positive (ie, does not induce symptoms). In other embodiments, the fibrosis is associated with pathological conditions that may or may not be reversible. In some embodiments, the fibrosis is liver fibrosis, pulmonary fibrosis, kidney fibrosis, bone marrow fibrosis, pancreatic fibrosis, skin fibrosis, cardiac fibrosis, arterial fibrosis, joint fibrosis, breast fibrosis. muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis and pleural fibrosis. In one embodiment, the fibrosis is liver fibrosis or pulmonary fibrosis. In other embodiments, the fibrosis is associated with tumors derived from cancer such as liver cancer, lung cancer, kidney cancer, breast cancer and/or pancreatic cancer. In one embodiment, the method reduces, reverses, alleviates, ameliorate, inhibits or delays or prevents the fibrosis, the fibrosis-related symptoms, the underlying cause of the fibrosis, or a combination thereof.

In other embodiments, the AAV vectors disclosed herein can treat, reduce, reverse, prevent, ameliorate, control or inhibit a fibrosis-related disease or disorder in a subject in need thereof. The term "disease- or disorder-related fibrosis" refers to fibrosis that accompanies, is caused by, or results from a disease or disorder. The term includes fibrosis caused by or caused by the diseases or disorders disclosed above or fibrosis associated with the disease or disorder.

In other embodiments, the AAV vectors of the present disclosure are capable of treating, reducing, reversing, preventing, delaying, ameliorating, controlling or inhibiting liver, lung, kidney, breast and/or pancreatic cancer in a subject in need thereof; The method includes administering an AAV vector to said subject. In some embodiments, the liver cancer, lung cancer, kidney cancer, breast cancer, and/or pancreatic cancer is associated with or caused by fibrosis.

In some embodiments, a therapeutically effective amount of an AAV vector or composition thereof of this disclosure is administered. When treating a subject (eg, a human), therapeutically effective amounts of the AAV vectors disclosed herein will vary according to factors such as age, sex, and severity of disease.

In some embodiments, AAV vectors or compositions thereof of the present disclosure are administered intravenously, orally, parenterally, intrathecally, intrathecally, intracerebroventricularly, intramuscularly, pulmonary, intraperitoneally, intravitreally, subcutaneously, It is administered epidurally, subretinally or intraventricularly.

In some embodiments, the AAV vectors or compositions thereof disclosed herein are combined with one or more additional agents to treat fibrosis (e.g., for idiopathic pulmonary fibrosis, pirfenidone (ESBRIET Registered (trademark)) or nintedanib (OFEV®)). Dosages and administration of the one or more additional therapeutic agents are known in the art, eg, as indicated on the product label of the respective drug.

In some embodiments, AAV vectors or compositions thereof described herein are used to treat central nervous system injury (e.g., traumatic brain injury, cerebrospinal injury, stroke or brain tumor), cerebrospinal injury, degenerative brain injury. administered in combination with one or more additional agents to treat disorders (e.g., Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ALS), degenerative cerebrospinal or neuropathy or neuropathic pain). obtain. For example, non-limiting exemplary formulations for treating Huntington's disease include tetrabenazine (XENAZINE®), such as haloperidol (HALDOL®), chlorpromazine, risperidone (RISPERDAL®) and quetiapine. including antipsychotic drugs such as (SEROQLIEL®).

Non-limiting examples of Parkinson's disease therapeutic agents include levodopa (with or without carbidopa), dopaminergic agents such as pramipexole (MIRAPEX®), ropinirole (REQETIP®) and rotigotine (NEEIPRO®). trademark)) and apomorphine (APOKYN®), selegiline (ELDEPRYL® and ZELAPAR®), rasagiline (AZILECT®), entacapone (COMTAN®), benztropine (COGENTIN ®), trihexyphenidyl and amantadine.

Non-limiting examples of Alzheimer's disease therapeutic agents include donepezil (ARICEPT®), galantamine (RAZADYNE®) and rivastigmine (EXELON®).

Non-limiting examples of formulations for treating multiple sclerosis include glatiramer acetate (COPAXONE®), dimethyl fumarate (TECFIDERA®), fingolimod (GILENYA®), teriflunomide (AEIBAGIO ( ®)), natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®) and mitoxantrone.

A non-limiting example of a formulation for treating ALS includes riluzole (RILETTEK®).

Dosages and administration of the one or more additional therapeutic agents are known in the art, eg, as indicated on the product label of the respective drug.

The examples described below are provided as non-limiting illustrations.

(Example 1: Adeno-associated virus (AAV) vector production)
AAV vectors encoding single chain variable fragments (scFv) of anti-FAM19A5 antibody clones 1-65, 2-13 or 3-2 were generated as follows. Briefly, the pscAAV-MCS expression vector (Cell biolabs, cat. #VPK-430) was cut with EcoRI and NotI to generate the AAV vector encoding the 3-2 scFv. Next, as shown in FIG. 1, a transgene encoding human beta-globin intron and 3-2 scFv under the CMV promoter was inserted into the pscAAV-MCS vector. A human beta globin intron was inserted for maximization of gene expression. To generate AAV vectors encoding 1-65 or 2-13 scFv, the above AAV vectors were stripped of the 3-2 scFv transgene using EcoRV and Notl. Transgenes encoding 1-65 or 2-13 ScFv were then inserted into AAV vectors. Transgenes encoding 3-2, 1-65 or 2-13 ScFv were codon optimized for optimal expression in mice. All AAV vectors contained a mouse IgG kappa chain signal peptide in front of the transgene to facilitate secretion from the cells. The AAV vector also contained a FLAG-tag after the scFv transgene to aid in gene expression detection. Once constructed, the AAV vector was transformed into E. coli, cultured, and maxiprep was used to obtain large quantities of the AAV vector. Signagen Laboratories (Rockville, Md.) generated and provided AAV vectors encoding 1-30 scFv or murine IL-10 or GFP.

The following recombinant AAVs were used in the examples described below: (i) scAAV8-CMV-3-2-ScFv (Sirion, Germany), (ii) scAAV9-CMV-3-2-ScFv (Signagen Laboratories, (iii) scAAV9-CMV-1-65-ScFv (Signagen Laboratories, USA), scAAV9-CMV-2-13-ScFv (Signagen Laboratories, USA); (iv) scAAV1-CMV-3-2-ScFv (Signagen Laboratories, USA); (v) scAAV9-CMV-1-30-ScFv (Signagen Laboratories, USA); (vi) scAAV9-CMV-mIL-10 (Signagen Laboratories, USA); (vii) scAAV9-CMV- GFP (Signagen Laboratories, USA); (viii) scAAV1-CMV-GFP (Signagen Laboratories, USA).

(Example 2: Anti-FAM19A5ScFv gene expression analysis)
Using the AAV vectors prepared above (scAAV8-CMV-3-2-ScFv, scAAV9-CMV-3-2-ScFv, scAAV9-CMV-1-65-ScFv and scAAV9-CMV-2-13-ScFv) , tested the expression of anti-FAM19A5 ScFv. Briefly, HEK293 cells (1.2×10 ⁶ ) were seeded in 60 mm ² dishes and cultured for 24 hours. The medium was then replaced with opti-MEM medium (low serum) and HEK293 cells were transduced with different AAV vectors (using multiplicity of infection (MOI)). Supernatants were harvested on days 1, 4 and 8 post-transduction. To confirm gene expression, the FLAG tag included after the scFv gene was detected using the ABSbio ^™ DYKDDDK tag ELISA kit (Advanced Bioreagent, cat.# SE002-flag) (see Figure 1 and Example 1). After diluting the recovered supernatant 1- to 10-fold, 100 μL of the diluted supernatant was added to the wells of the ELISA plate and reacted at room temperature at 350 rpm for 2 hours. The plates were then washed and 100 μL of detection antibody was added to each well and allowed to react for 1 hour at 350 rpm and room temperature. The plate was then washed again and 100 μL of TMB solution was added to each well and left for 10-30 minutes. 100 μL of stop solution was then added to the wells and the absorbance was measured at 450 nm. The amount of FLAG was measured from the standard curve and the amount of scFv was calculated by multiplying the FLAG expression ratio to the molecular weight of the standard.

As shown in FIG. 2A, with the scAAV8-CMV-3-2-scFv vector, anti-FAM19A5 scFv could be detected in the supernatant as early as day 1 after transduction (1×10 ⁵ MOI). Approximately 100 ng/mL of 3-2 scFv was detected in the supernatant by 8 days post-transduction (at 1×10 ⁵ MOI). Approximately 70 ng/mL of 3-2 scFv was detected in the supernatant by 8 days post-transduction with the scAAV9-CMV-3-2-scFv vector (2×10 ⁵ MOI). Similar results were observed with scAAV9-CMV-1-65-ScFv and scAAV9-CMV-2-13-ScFv. As shown in FIG. 2C, approximately 120 ng/mL and 115 ng/mL of 1-65 scFv and 2-13 scFv, respectively, were detected in the supernatant 4 days after transduction.

The data demonstrate that all AAV vectors in Example 1 were successfully engineered and that they are capable of inducing anti-FAM19A5 scFv expression upon transduction into cells.

(Example 3: Suppression of FAM19A5 binding in U87MG cells)
To assess the binding capacity of the anti-FAM19A5 scFv produced by the AAV constructs, binding inhibition assays were performed using U87MG cells, a human primary glioblastoma cell line capable of binding to human FAM19A5-Fc protein. gone. Briefly, U87MG cells were treated with scAAV8-CMV-2-3-ScFv (MOI 1 x ¹⁰⁵ ), scAAV9-CMV-1-65-ScFv (MOI 2 x ¹⁰⁵ ) or scAAV9-CMV-2-13-ScFv (MOI 2 x 105). MOI 2×10 ⁵ ). Then, 4 days after transduction, 0.1 μg of human FAM19A5-Fc protein was mixed with 200 μL of supernatant and the mixture was incubated at 4° C. for 30 minutes. Negative controls were supernatants of HEK293 cells transduced with PBS or scAAV9-CMV-GFP vector. After 30 minutes of incubation, 100 μL of the mixture (supernatant of cells transduced with human FAM19A5-Fc + anti-FAM19A5 scFv AAV construct) was added to U87MG cells (2×10 ⁵ ) and reacted at 4° C. for 1 hour. let me The cells were then washed with PBS, treated with 100 μL of sheep anti-human IgG488 antibody (Invitrogen, cat.# A11013) (diluted 1:50) and further incubated at 4° C. for 30 minutes. The cells were then washed again and binding of human FAM19A5-Fc to U87MG cells was analyzed using flow cell counting.

As shown in Figures 3A, 3B and 3C, when the U87MG cells were treated with the mixture (supernatant of cells transfected with human FAM19A5-Fc + anti-FAM19A5 scFv AAV construct), Binding of human FAM19A5-Fc was reduced when the cells were treated with the control mixture (supernatant of cells transfected with human FAM19A5-Fc + scAAV9-CMV-GFP construct). This is the case for all anti-FAM19A5 scFv AAV constructs tested. Supernatants of cells transduced with scAAV9-CMV-1-65-ScFv, scAAV9-CMV-2-13-ScFv, scAAV8-CMV-3-2-ScFv were added to the appropriate control group (AAV- The mean fluorescence intensity (MFI) of binding of human FAM19A5-Fc to U87MG cells decreased by 19% (FIG. 3A), respectively, compared to the supernatant of cells transfected with GFP vector or PBS-treated). 68% (Fig. 3B) and 42% (Fig. 3C) reduction.

These results demonstrate that the anti-FAM19A5 scFv produced by the AAV constructs described in Example 1 can effectively bind to human FAM19A5 with high affinity.

(Example 4: Inhibition of FAM19A5 binding in C8-D1A cells)
To confirm the binding inhibition observed in Example 3, C8-D1A cells were used for binding inhibition assays. C8-D1A cells are an astrocyte cell line and can secrete FAM19A5 into the surrounding medium when cultured. If the anti-FAM19A5 scFv produced by said AAV construct can effectively bind to human FAM19A5, add C8-D1A cell culture medium with the supernatant of cells transduced with said anti-FAM19A5 scFv AAV. Upon treatment, a decrease in FAM19A5 protein concentration within the culture medium should be seen.

Briefly, C8-D1A cells were plated in 6-well plates (2×10 ⁵ cells/well) and cultured for approximately 24 hours. Anti-FAM19A5 scFv-containing supernatants induced HEK293 cells to: (i) scAAV8-CMV-3-2-ScFv (MOI 1 x 10 ⁵ ); (ii) scAAV9-CMV-1-65-ScFv (MOI = 2 x 10 or ( ⁱⁱⁱ ) by transfection with either one of scAAV9-CMV-2-13-ScFv (MOI=2×10 ⁵ ), followed by supernatant 8 days post-transduction. recovered. To activate the C8-D1A cells, TGF-β (20 ng/mL) was added to the cells along with supernatant containing anti-FAM19A5 scFv. Supernatants of cells transduced with PBS or scAAV-9-CMV-GFP were used as negative controls. After culturing the cells for 24 hours, ELISA analysis was used to measure FAM19A5 protein concentration in the cultures (see Example 1 for the method used for ELISA analysis).

As shown in FIG. 4A, when C8-D1A cells were activated in the presence of supernatant of cells transduced with scAAV8-CMV-3-2-ScFv, the corresponding control group ( Approximately 38% reduction in the concentration of FAM19A5 protein detected from the culture medium compared to PBS). Supernatants of cells transduced with scAAV9-CMV-2-13-ScFv and scAAV9-CMV-1-65-ScFv were added to the appropriate control group (of cells transduced with the GFP AAV vector). The concentration of FAM19A5 protein in the culture medium decreased by approximately 63% and 14%, respectively, compared to the supernatant). See FIG. 4B.

The above data further demonstrate that the anti-FAM19A5 scFv produced by the AAV vector can effectively bind to human FAM19A5.

(Example 5: In vivo expression analysis)
To confirm in vivo anti-FAM19A5 scFv expression, the scAAV8-CMV-3-2-ScFv construct was administered intrathecally to C57BL/6 mice (1×10 ¹¹ viral genomes (vg)/mouse). The mice were then sacrificed 1 and 2 weeks after dosing and the spinal cord tissue (lumbar spinal cord) was analyzed. Anti-FLAG antibody and fluorescence microscopy were used to detect gene expression.

As shown in FIG. 5, naive animals (i.e., not administered AAV vectors) did not develop positive antibody expression at 1 week (upper left photo) and 2 weeks (lower left photo) after dosing. . In contrast, in scAAV8-CMV-3-2-ScFv-treated animals, anti-FLAG (ie, anti-FAM19A5 scFv) expression was observed in ventral horn neurons 1 week after administration (upper right photograph). ). By 2 weeks there was much higher expression, including in the dorsal horn region.

This data supports the above in vitro expression data (see Example 2) and implies that upon intrathecal administration of the anti-FAM19A5 AAV vector, surrounding cells can be transduced to produce anti-FAM19A5 scFv.

(Example 6: Evaluation of pain relief in a chronic neuropathic pain model (CCI) using scAAV8-CMV-3-2-ScFv)
A mouse model of chronic contractile injury (CCI) was used to evaluate the in vivo effects of administration of the anti-FAM19A5 scFv AAV construct. Briefly, PBS (control group) or scAAV8-CMV-3-2-ScFv were administered intrathecally (into the spine) to C57BL/6 mice. Approximately one week after dosing, peripheral nerve injury was then induced from the animals by sciatic nerve ligation. At various time points after injury, the animals were assessed for both mechanical allodynia (response to external physical stimuli) and thermal hyperalgesia (response to high temperature). Mechanical allodynia was assessed by applying Von Frey monofilament (0.16 g) to the injured paw several times and observing the number of times the animal responded with pain. Thermal hyperalgesia was assessed using the Hargreaves test, in which a radiant heat stimulus (intensity: 30) was applied to the injured paw and paw withdrawal latencies were determined.

As shown in Figure 6B, animals receiving scAAV8-CMV-3-2 responded less frequently to Von Frey monofilaments than control animals (PBS only). Similar results were observed for thermal hyperalgesia. See FIG. 6B. These results demonstrate that in vivo administration of anti-FAM19A5AAV constructs can reduce neuropathic pain after peripheral nerve injury.

(Example 7: Evaluation of pain relief in a chronic neuropathic pain model (CCI) using scAAV9-CMV-3-2-ScFv)
The in vivo efficacy of scAAV9-CMV-3-2-ScFv was also tested using the mouse model of chronic contractile injury (CCI) described in Example 6. Briefly, the scAAV9-CMV-3-2-ScFv construct was administered intrathecally to C57BL6 mice. Control animals received the scAAV9-CMV-GFP construct. Peripheral nerve injury was then induced approximately one week after administration. The animals were then evaluated for both mechanical allodynia and thermal hyperalgesia at various time points, as described in Example 6.

As shown in Figures 7A and 7B, animals administered scAAV9-CMV-3-2-ScFv appeared to have more pain tolerance after peripheral nerve injury. For example, compared to control animals, scAAV9-CMV-3-2-ScFv-treated mice responded less frequently to Von Frey monofilaments (Fig. 7A) and had a higher latency time to high temperature. (Fig. 7B).

Altogether, these results demonstrate that anti-FAM19A5 scFv can be effectively delivered in vivo using AAV vectors, and that anti-FAM19A5 scFv produced by AAV vectors are fully functional (e.g., bind human FAM19A5 and bind to neurons). pathological pain).

(Example 8: Assessment of pain relief in chronic neuropathic pain model (CCI) using scAAV1-CMV-3-2-ScFv)
To further assess whether the effect depends on the type of AAV used, the in vivo effect of scAAV1-CMV-3-2-ScFv was tested. Briefly, C57BL/6 mice were administered two doses of scAAV1-CMV-3-2-ScFv (each dose=1×10 ¹¹ vg/mouse) at 2-week intervals by intrathecal administration. One week after the last dose, peripheral nerve injury was induced and both mechanical allodynia and passionate hyperalgesia were assessed as described in Example 6.

As observed from Examples 6 and 7, animals treated with scAAV1-CMV-3-2-ScFv were more susceptible to hemorrhoids from approximately 4 days after injury induction (i.e., several days after surgery) than the control group. Showed improvement in mechanical heteropathic pain (ie, responding less frequently to Von Frey monofilament stimulation). See FIG. 8A. These improvements were maintained until the end of the experiment 24 days after injury induction. In the case of thermal hyperalgesia, an improvement was observed in animals treated with scAAV1-CMV-3-2-ScFv from about 5 days after injury induction (ie, longer latency to respond to thermal stimuli), at least after injury induction. It lasted up to 12 days. See FIG. 8B.

These results further support the efficacy of 3-2 anti-FAM19A5 scFv in treating neuropathic pain and demonstrate that therapeutic benefit is independent of the type of AAV vector used.

Example 9: Pain relief assessment in chronic neuropathic pain model (CCI) using scAAV9-CMV-2-13-ScFv or scAAV9-CMV-1-30-ScFv
To assess whether other clones of the anti-FAM19A5 antibody can also treat neuropathic pain, the mouse model of chronic contractile injury (CCI) described in Example 6 was again used. Briefly, scAAV9-CMV-2-13-ScFv (2 doses 2 weeks apart, each dose = 1 x 10 ¹¹ vg/mouse) or scAAV9-CMV-1-30-ScFv (single dose, 1 x 10 ¹¹ vg/mouse) was administered to C57BL/6 mice by intrathecal administration. Control animals received scAAV9-CMV-GFP. Peripheral nerve injury was induced approximately one week after the last dose, and both mechanical allogenic pain and thermal hyperalgesia were assessed at various time points after injury induction (see Example 6).

As shown in FIGS. 9A, 9B, 10A, and 10B, animals treated with scAAV9-CMV-2-13-ScFv or scAAV9-CMV-1-30-ScFv exhibited mechanical allodynia and thermal hyperalgesia. showed improvement for both. These results demonstrate that individual clones of anti-FAM19A5 antibodies can also be used to treat neuropathic pain.

Example 10: Evaluation of pain relief in a chronic neuropathic pain model (CCI) using a combination of anti-FAM19A5ScFv and IL-10
To determine the efficacy of anti-FAM19A5 ScFv in combination with other therapeutic agents (ie, IL-10), one of the following regimens was administered to C57BL/6 mice by intrathecal administration: (i) scAAV1-CMV-GFP; (ii) scAAV1-CMV-3-2-ScFv; (iii) scAAV1-CMV-3-2-ScFv+scAAV9-CMV-mIL-10; and (iv) scAAV9-CMV-mIL -10. The different treatment regimens described above were administered to mice twice (2 weeks apart) at a dose of 1×10 ¹¹ μg/mouse. Peripheral nerve injury was induced one week after the last dose and mechanical allogenic pain was assessed at various time points (see Example 6).

As observed in the previous example, animals treated with scAAV1-CMV-3-2-ScFv alone were more resistant to physical stimulation than the control group (i.e., scAAV1-CMV-GFP). (ie, responds to the application of the Von Frey monofilament relatively infrequently). A similar response was observed from animals treated with scAAV9-CMV-mIL-10 alone (FIG. 11A). When animals were treated with the combination of scAAV1-CMV-3-2-ScFv and scAAV9-CMV-mIL-10, they showed a much improved response compared to animals treated with a single treatment regimen. Similar results were observed for scAAV9-CMV-2-13-ScFv (FIG. 11B) and scAAV9-CMV-1-30-ScFv (FIG. 11C).

These results further demonstrate the efficacy of anti-FAM19A5 scFv delivered through AAV vectors in neuropathic pain therapy. These results also demonstrate that this efficacy can be enhanced when combined with other therapeutic agents such as IL-10.

This PCT application claims the priority benefit of US Provisional Application No. 62/668,634, filed May 8, 2018, which is hereby incorporated by reference in its entirety.

Figure 1 provides a map of the pscAAV constructs used to generate the anti-FAM19A5 scFv AAV constructs described in the Examples. As shown in Figure 1, the pscAAV construct contains the following core elements: (i) human beta globin under the CMV promoter; (ii) encoding 3-2, 1-65 or 2-13 scFv. and (iii) left-right inverted terminal repeats (ITRs). Figures 2A, 2B and 2C show the expression levels of anti-FAM19A5 scFv after transduction of HEK293 cells with any one of the following AAV constructs: (i) scAAV8-CMV- (ii) scAAV9-CMV-3-2-ScFv; (iii) scAAV9-CMV-1-65-ScFv; or (iv) scAAV9-2-13-ScFv. In FIG. 2A, HEK293 cells were transduced with scAAV8-CMV-3-2-ScFv at two different MOIs: 5×10 ⁴ (white bars) or 1×10 ⁵ (black bars). Antibody concentrations were measured 1, 4 and 8 days after transduction. In FIG. 2B, HEK293 cells were transduced with scAAV9-CMV-3-2-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ). Antibody concentrations were measured at a single time point, 8 days after transduction. In FIG. 2C, HEK293 cells were transfected with scAAV9-CMV-1-65-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ) or scAAV9-2-13-ScFv (white bars with dots: MOI=1×10 ⁵ ; black bars with dots=2×10 ⁵ ). Figures 2A, 2B and 2C show the expression levels of anti-FAM19A5 scFv after transduction of HEK293 cells with any one of the following AAV constructs: (i) scAAV8-CMV- (ii) scAAV9-CMV-3-2-ScFv; (iii) scAAV9-CMV-1-65-ScFv; or (iv) scAAV9-2-13-ScFv. In FIG. 2A, HEK293 cells were transduced with scAAV8-CMV-3-2-ScFv at two different MOIs: 5×10 ⁴ (white bars) or 1×10 ⁵ (black bars). Antibody concentrations were measured 1, 4 and 8 days after transduction. In FIG. 2B, HEK293 cells were transduced with scAAV9-CMV-3-2-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ). Antibody concentrations were measured at a single time point, 8 days after transduction. In FIG. 2C, HEK293 cells were transfected with scAAV9-CMV-1-65-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ) or scAAV9-2-13-ScFv (white bars with dots: MOI=1×10 ⁵ ; black bars with dots=2×10 ⁵ ). Figures 2A, 2B and 2C show the expression levels of anti-FAM19A5 scFv after transduction of HEK293 cells with any one of the following AAV constructs: (i) scAAV8-CMV- (ii) scAAV9-CMV-3-2-ScFv; (iii) scAAV9-CMV-1-65-ScFv; or (iv) scAAV9-2-13-ScFv. In FIG. 2A, HEK293 cells were transduced with scAAV8-CMV-3-2-ScFv at two different MOIs: 5×10 ⁴ (white bars) or 1×10 ⁵ (black bars). Antibody concentrations were measured 1, 4 and 8 days after transduction. In FIG. 2B, HEK293 cells were transduced with scAAV9-CMV-3-2-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ). Antibody concentrations were measured at a single time point, 8 days after transduction. In FIG. 2C, HEK293 cells were transfected with scAAV9-CMV-1-65-ScFv (white bars: MOI=1×10 ⁵ ; black bars=2×10 ⁵ ) or scAAV9-2-13-ScFv (white bars with dots: MOI=1×10 ⁵ ; black bars with dots=2×10 ⁵ ). Figures 3A, 3B and 3C show the mean fluorescence intensity (MFI) of human FAM19A5-Fc binding to U87MG cells in the presence or absence of supernatants of cells transduced with different anti-FAM19A5AAV constructs. ing. FIG. 3A shows data for scAAV9-CMV-1-65-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 1-65scFv. FIG. 3B shows data for scAAV9-2-13-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 2-13 scFv. FIG. 3C shows data for scAAV8-CMV-3-2-ScFv. The number "1" indicates the control group (PBS) and the number "2" indicates the 3-2 scFv. Figures 3A, 3B and 3C show the mean fluorescence intensity (MFI) of human FAM19A5-Fc binding to U87MG cells in the presence or absence of supernatants of cells transduced with different anti-FAM19A5AAV constructs. ing. FIG. 3A shows data for scAAV9-CMV-1-65-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 1-65scFv. FIG. 3B shows data for scAAV9-2-13-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 2-13 scFv. FIG. 3C shows data for scAAV8-CMV-3-2-ScFv. The number "1" indicates the control group (PBS) and the number "2" indicates the 3-2 scFv. Figures 3A, 3B and 3C show the mean fluorescence intensity (MFI) of human FAM19A5-Fc binding to U87MG cells in the presence or absence of supernatants of cells transduced with different anti-FAM19A5AAV constructs. ing. FIG. 3A shows data for scAAV9-CMV-1-65-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 1-65scFv. FIG. 3B shows data for scAAV9-2-13-ScFv. The number "1" indicates the control group (GFP) and the number "2" indicates the 2-13 scFv. FIG. 3C shows data for scAAV8-CMV-3-2-ScFv. The number "1" indicates the control group (PBS) and the number "2" indicates the 3-2 scFv. Figures 4A and 4B show the concentration of human FAM19A5 protein in C8-D1A cell cultures after treatment with supernatants of cells transduced with different anti-FAM19A5AAV constructs. FIG. 4A compares FAM19A5 protein concentration when C8-D1A cells were cultured with the supernatant of cells transduced with scAAV8-CMV-3-2-ScFv or with PBS alone. FIG. 4B compares FAM19A5 concentration when C8-D1A cells were cultured with scAAV9-2-13-ScFv, scAAV9-CMV-1-65-ScFv or control construct (scAAV9-CMV-GFP). . In Figures 4A and 4B, "naive" represents C8-D1A cells that are not activated and produce minimal FAM19A5 protein in culture. Figures 4A and 4B show the concentration of human FAM19A5 protein in C8-D1A cell cultures after treatment with supernatants of cells transduced with different anti-FAM19A5AAV constructs. FIG. 4A compares FAM19A5 protein concentration when C8-D1A cells were cultured with the supernatant of cells transduced with scAAV8-CMV-3-2-ScFv or with PBS alone. FIG. 4B compares FAM19A5 concentration when C8-D1A cells were cultured with scAAV9-2-13-ScFv, scAAV9-CMV-1-65-ScFv or control construct (scAAV9-CMV-GFP). . In Figures 4A and 4B, "naive" represents C8-D1A cells that are not activated and produce minimal FAM19A5 protein in culture. Figure 5 shows intracerebral anti-FAM19A5(3-2) scFv levels 1 week (upper row) and 2 weeks (lower row) after intrathecal administration of scAAV8-CMV-3-2-ScFv to C57BL/6 mice. Expression is shown. Photographs in the left column are of untreated animals (untreated). Photographs in the right column are of mice administered the AAV constructs. In the top right row of photographs, arrows denote positive staining. Figures 6A and 6B show the effects of scAAV8-CMV-3-2-ScFv administration on mechanical allodynia (Figure 6A) and thermal hyperalgesia (Figure 6B) after peripheral nerve injury. In FIG. 6A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 6B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 6A and 6B show the effects of scAAV8-CMV-3-2-ScFv administration on mechanical allodynia (Figure 6A) and thermal hyperalgesia (Figure 6B) after peripheral nerve injury. In FIG. 6A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 6B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 7A and 7B show the effects of scAAV9-CMV-3-2-ScFv administration on mechanical allodynia (Figure 7A) and thermal hyperalgesia (Figure 7B) after peripheral nerve injury. In FIG. 7A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 7B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 7A and 7B show the effects of scAAV9-CMV-3-2-ScFv administration on mechanical allodynia (Figure 7A) and thermal hyperalgesia (Figure 7B) after peripheral nerve injury. In FIG. 7A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain to stimulation with Von Frey microfilaments. In FIG. 7B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 8A and 8B show the effects of scAAV1-CMV-3-2-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 8A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 8B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 8A and 8B show the effects of scAAV1-CMV-3-2-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 8A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 8B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 9A and 9B show the effects of scAAV9-CMV-2-13-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 9A, the effect on mechanical allogenic pain is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In Figure 9B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 9A and 9B show the effects of scAAV9-CMV-2-13-ScFv administration on mechanical allogenic pain and thermal hyperalgesia, respectively. In FIG. 9A, the effect on mechanical allodynia is expressed as the number of times the animal responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 9B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 10A and 10B show the effects of scAAV9-CMV-1-30-ScFv administration on mechanical allodynia and thermal hyperalgesia, respectively. In FIG. 10A, the effect on mechanical allodynia is expressed as the number of times animals responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 10B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 10A and 10B show the effects of scAAV9-CMV-1-30-ScFv administration on mechanical allodynia and thermal hyperalgesia, respectively. In FIG. 10A, the effect on mechanical allodynia is expressed as the number of times animals responded to pain (out of 10 stimuli) to stimulation with Von Frey microfilaments. In FIG. 10B, the effect on thermal hyperalgesia is displayed as paw withdrawal latency (the time it took the animal to leave the heat source). "*" denotes statistical significance of p<0.05 vs control group. Figures 11A, 11B and 11C provide comparative effects of different anti-FAM19A5 scFv AAV constructs alone or in combination with other therapeutic agents. In FIG. 11A, animals were administered one of the following: (i) scAAV1-CMV-GFP (open circles); (ii) scAAV1-CMV-3-2-ScFv (filled circles); (iii). scAAV9-CMV-mIL-10 (open squares); (iv) scAAV1-CMV-3-2-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11B, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-2-13-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-2-13-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11C, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-1-30-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-1-30-ScFv+scAAV9-CMV-mIL-10 (filled squares). "*" denotes statistical significance of p<0.05 vs control group. Figures 11A, 11B and 11C provide comparative effects of different anti-FAM19A5 scFv AAV constructs alone or in combination with other therapeutic agents. In FIG. 11A, animals were administered one of the following: (i) scAAV1-CMV-GFP (open circles); (ii) scAAV1-CMV-3-2-ScFv (filled circles); (iii). scAAV9-CMV-mIL-10 (open squares); (iv) scAAV1-CMV-3-2-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11B, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-2-13-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-2-13-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11C, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-1-30-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-1-30-ScFv+scAAV9-CMV-mIL-10 (filled squares). "*" denotes statistical significance of p<0.05 vs control group. Figures 11A, 11B and 11C provide comparative effects of different anti-FAM19A5 scFv AAV constructs alone or in combination with other therapeutic agents. In FIG. 11A, animals were administered one of the following: (i) scAAV1-CMV-GFP (open circles); (ii) scAAV1-CMV-3-2-ScFv (filled circles); (iii). scAAV9-CMV-mIL-10 (open squares); (iv) scAAV1-CMV-3-2-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11B, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-2-13-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-2-13-ScFv+scAAV9-CMV-mIL-10 (filled squares). In FIG. 11C, animals were administered either: (i) scAAV9-CMV-GFP (open circles); (ii) scAAV9-CMV-1-30-ScFv (filled circles); (iii) scAAV9. -CMV-mIL-10 (open squares); (iv) scAAV9-CMV-1-30-ScFv+scAAV9-CMV-mIL-10 (filled squares). "*" denotes statistical significance of p<0.05 vs control group.

Claims (23)

A composition for treating neuropathic pain in a subject in need thereof, comprising:
said composition comprising an adeno-associated virus (AAV) vector;
said AAV vector comprises a nucleic acid encoding an antagonist to a Family, member A5 (FAM19A5) protein with sequence similarity 19;
A composition , wherein said FAM19A5 antagonist is a monoclonal antibody or antigen-binding fragment thereof (anti-FAM19A5 antibody) that specifically binds to said FAM19A5 protein. 2. The composition of claim 1, further comprising an intron, a signal peptide and/or one or more adeno-associated virus inverted terminal repeats (ITRs). 2. The composition of claim 1, wherein said anti-FAM19A5 antibody comprises Fab, Fab', F(ab')2, Fv or single chain Fv (scFv). 4. The composition of claim 3, wherein said anti-FAM19A5 antibody is a scFv. The composition of any of claims 1-4, wherein said nucleic acid is codon optimized. 6. The composition of any of claims 1-5, wherein said nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206 or SEQ ID NO:301. 5. The composition of claim 4, wherein said scFv comprises the amino acid sequence set forth in SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203 or SEQ ID NO:256. wherein said anti-FAM19A5 antibody:
(a) the property of binding soluble human FAM19A5 with a K _D of 10 nM or less as measured by an enzyme-linked immunosorbent assay (ELISA);
(b) the property of binding to membrane-bound human FAM19A5 with a _KD of 10 nM or less when measured by ELISA; or (c) both (a) and (b). 8. The composition according to any one of -7. said anti-FAM19A5 antibody comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3;
(i) said heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOS: 17, 18 and 19 respectively, and said light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOS: 29, 30 and 31 respectively;
(ii) said heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 14, 15 and 16, respectively, and said light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 26, 27 and 28, respectively;
(iii) said heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOS: 11, 12 and 13 respectively and said light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOS: 23, 24 and 25 respectively; or (iv) 9. Any of claims 1-8, wherein the heavy chain CDRl, CDR2 and CDR3 comprise SEQ ID NOs: 212, 213 and 16, respectively, and the light chain CDRl, CDR2 and CDR3 comprise SEQ ID NOs: 222, 225 and 224, respectively. The composition according to . 10. The composition of claim 9, wherein said anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:37 and a light chain variable domain comprising SEQ ID NO:41. 10. The composition of claim 9, wherein said anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:36 and a light chain variable domain comprising SEQ ID NO:40. 10. The composition of claim 9, wherein said anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:35 and a light chain variable domain comprising SEQ ID NO:39. 10. The composition of claim 9, wherein said anti-FAM19A5 antibody comprises a heavy chain variable domain comprising SEQ ID NO:236 and a light chain variable domain comprising SEQ ID NO:245. said anti-FAM19A5 antibody is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% binding of the reference antibody to human FAM19A5 as measured by a binding inhibition assay or 100% inhibition . The anti-FAM19A5 antibody is at least 10%, 20%, 30%, 40%, 50%, 60% when the concentration of FAM19A5 protein in culture medium containing an astrocyte cell line (e.g., C8-D1A) is measured by ELISA. %, 70%, 80 % , 90% or 100%. 1. A method of making a composition for treating neuropathic pain in a subject in need thereof, comprising:
said composition comprising recombinant adeno-associated virus (AAV) particles;
(a) culturing a host cell transfected with said AAV vector as defined in any one of claims 1 to 15 to provide a cell culture; A method of production comprising recovering the recombinant AAV particles from the supernatant. 16. The composition of any one of claims 1-15 for in vivo delivery of an antagonist to the Family, member A5 (FAM19A5) protein with sequence similarity 19 ("FAM19A5 antagonist"). A composition according to any of claims 1-15 and 17 for use wherein said neuropathic pain is peripheral neuropathic pain. A composition according to any one of claims 1 to 15 for increasing the threshold or latency to external stimuli in a subject in need thereof. 20. A composition according to claim 19 for applications wherein said external stimulus is mechanical stimulus. 20. A composition according to claim 19, for use in which the external stimulus is thermal stimulus. Uses in which a composition comprising the AAV vector is administered intravenously, orally, parenterally, intrathecally, intracerebroventricularly, pulmonary, intramuscularly, subcutaneously, intraperitoneally, intravitreally, epidurally, subretinally or intraventricularly The composition according to any one of claims 18-21, for A composition according to any of claims 18-22 for use wherein said subject is a human.

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